Methods and compositions for light-controllable virus production

Abstract

Provided herein are methods and systems for producing viral proteins and viral particles. In some cases, the methods and systems involve the use of optical stimulation for inducing expression of one or more viral proteins. In some cases, the methods and systems involve the use of optical switches for producing one or more viral proteins.

Description

WSGR Docket No. 60885-743.601METHODS AND COMPOSITIONS FOR LIGHT-CONTROLLABLE VIRUS PRODUCTION CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 738,450, filed December 23, 2024, which is entirely incorporated herein by reference.BACKGROUND

[0002] Recombinant adeno-associated viruses (rAAVs) are among the most widely used vectors for gene therapies. rAAVs are modified forms of naturally occurring adeno-associated viruses (AAVs) that contain therapeutic payloads. For example, rAAVs can be delivered intravenously in a tissue specific manner. However, current methods of producing rAAVs involve several steps, are difficult to scale and do not allow for precise temporal or spatial control of AAV protein expression.SUMMARY

[0003] Provided herein is a method of producing at least one viral protein, the method comprising: (a) providing or obtaining a cell comprising at least one exogenous nucleic acid comprising at least one nucleic acid sequence operably linked to a promoter and encoding at least one viral protein, and (b) controlling expression of the at least one viral protein using light.

[0004] Provided herein is a cell comprising at least one exogenous nucleic acid comprising at least one nucleic acid sequence operably linked to a promoter and encoding at least one viral protein. In some embodiments, the cell comprises at least one nucleic acid sequence encoding at least one light-controllable transcriptional regulator or at least one nucleic acid sequence encoding thereof. In some embodiments, the at least one light-controllable transcriptional regulator is activatable, upon modulated exposure to a light, to induce expression of the at least one viral protein under the control of the promoter.

[0005] Provided herein is a cell culture comprising a plurality of the cell(s) described herein; and a cell culture media. In some embodiments, the plurality of the cells is in suspension. In some embodiments, provided herein is a bioreactor comprising the cell culture described herein.

[0006] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details areWSGR Docket No. 60885-743.601capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0007] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0009] FIGS. 1A and IB depict non-limiting examples of a system for controlling expression of one or more viral proteins using a light-dimerizing domain coupled to a transcriptional activator in accordance with embodiments herein.

[0010] FIGS. 2A and 2B depict non-limiting examples of a system for controlling expression of one or more viral proteins using a light-dissociating domain coupled to a transcriptional activator in accordance with embodiments herein.

[0011] FIGS. 3A and 3B depict non-limiting examples of a system for controlling expression of one or more viral proteins using a light-sensitive subcellular localization module coupled to a transcriptional activator in accordance with embodiments herein.

[0012] FIGS. 4A and 4B depict non-limiting examples of a system for controlling expression of one or more viral proteins using a light-dimerizing domain coupled to a transcriptional repressor in accordance with embodiments herein.

[0013] FIGS. 5A and 5B depict non-limiting examples of a system for controlling expression of one or more viral proteins using a light-dissociating domain coupled to a transcriptional repressor in accordance with embodiments herein.WSGR Docket No. 60885-743.601

[0014] FIGS. 6A and 6B depict non-limiting examples of a system for controlling expression of one or more viral proteins using a light-sensitive subcellular localization module coupled to a transcriptional repressor in accordance with embodiments herein.

[0015] FIG. 7 depicts a non-limiting schematic representation of a system for controlling expression of one or more viral proteins using a recombinase.

[0016] FIG. 8 depicts a non-limiting schematic representation of AAV production.

[0017] FIG. 9 depicts non-limiting example plasmids containing the expression cassettes for the optogenetic gene expression system for stable integration.

[0018] FIGs. 10A and 10B show example flow cytometry analysis of cell lines with stable integration of the optogenetic gene expression system (OptoChassis). FIG. 10A displays mScarlet and GFP fluorescence. FIG. 10B displays quantification of the percentage of light-responsive cells from FIG. 10A.

[0019] FIG. 11 depicts non-limiting example plasmids containing the light-inducible expression cassettes for the adenoviral helper proteins.

[0020] FIGs. 12A and 12B illustrate a non-limiting example of AAV vector production using light-controllable adenoviral helper protein expression in a transient transfection system. FIG.12A shows AAV potency by a transduction assay (top panel) and vg titer by ddPCR (lower panel).FIG. 12B displays Western Blot analysis of the production cells for E2A, Rep, and Cap proteins.

[0021] FIG. 13 illustrates a non-limiting example that temporal control of adenoviral helper protein expression enhances AAV titer compared to constitutive expression, as measured by transduction assay.

[0022] FIG. 14 depicts non-limiting example plasmids containing the light-inducible expression cassettes for the Rep proteins used for stable integration.

[0023] FIG. 15 illustrates example screening of clones for light-inducible Rep40 / 68 expression by Western Blotting.

[0024] FIGs. 16A-16C illustrate example characterization of cell viability and proliferation under dark and light conditions for clones with light-inducible Rep40 / 68 expression. FIG. 16A presents doubling time in the dark. FIG. 16B depicts cell viability. FIG. 16C illustrates cell proliferation.

[0025] FIGs. 17A and 17B illustrate example stable clones with light-inducible Rep40 / 68 expression enabling AAV vector production upon illumination. FIG. 17A shows AAV vector potency by a transduction assay. FIG. 17B shows Western Blot analysis for Rep proteins.

[0026] FIG. 18 illustrate an example of stable pools with light-inducible Rep52 / 78 expression enabling AAV vector production upon illumination, with potency measured by transduction assay.WSGR Docket No. 60885-743.601

[0027] FIG. 19 depicts non-limiting example plasmids containing the light-inducible expression cassettes for Rep proteins used for transient production.

[0028] FIG. 20 illustrates an example of light-controllable Rep protein expression in transient transfection enabling AAV vector production, with potency measured by transduction assay.

[0029] FIG.21 depicts example plasmids for production of GFP-encoding AAV vectors together with the light-inducible Rep and Helper constructs.DETAILED DESCRIPTION

[0030] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.I. Methods, Cells, Cell Cultures, and Bioreactors for Producing Viral Protein(s)

[0031] The methods and compositions described herein address major challenges in the production of recombinant adeno-associated viral vectors (rAAVs). Currently, AAVs are mainly produced by triple transfection of three plasmids into producer cells. This process does not scale very well due to the high amount of GMP -grade plasmid and transfection reagent required. Further, the complexity of this process and the requirement for multiple inputs (e.g., plasmids and transfection reagents) is inherently linked to batch-to-batch variations. Provided herein are optimized and superior approaches for the generation of stable packaging or producer cell lines for AAVs. The key challenge in the process is that the Rep proteins, large Rep proteins (e.g., Rep78 and Rep68) and small Rep proteins (e.g., Rep52 and Rep40), can be cytostatic and cytotoxic which requires a conditional expression only at the production state. Provided herein are methods and composition for producing a stable AAV producer line which uses light to control the production of at least one Rep protein (e.g., Rep78, Rep68, Rep52, or Rep40). In some embodiments, the methods and composition comprise producing a stable AAV producer line, which uses light to control the production of at least one Cap protein (e.g., VP1, VP2, or VP3).

[0032] The precise temporal control of viral protein expression described herein further improves the yield and quality (e.g., a ratio of full to empty capsids) of the produced AAV vectors. During the natural replication of AAVs the expression of the different viral proteins, in particular of the Rep and Cap proteins, is precisely controlled. For example, first the large Rep proteins involved in AAV genome replication are expressed followed by expression of the small Rep proteins involved in viral packaging. In contrast, in the traditional triple transfection approachesWSGR Docket No. 60885-743.601expression of all viral proteins is induced at exactly the same time, resulting in lower yield and quality of the produced AAV vectors. The methods and composition described herein allow precise and reversible control of the expression of the viral proteins using light and therefore allow the temporal dynamics of AAV replication to be mimicked in a bioproduction setting of AAV vectors resulting in higher quality and yield. The increased ratio of full to empty capsids addresses a major bottleneck in current AAV production systems which produce 50-99% empty capsids and therefore strongly increase downstream processing costs and side effects in patients.

[0033] Provided herein in some embodiments are methods and compositions for producing at least one viral protein. In some embodiments, the methods and compositions described herein comprises providing or obtaining a cell comprising at least one exogenous nucleic acid comprising at least one nucleic acid sequence operably linked to a promoter and encoding at least one viral protein. In some embodiments, the methods and compositions described herein comprises providing or obtaining a cell comprising at least one exogenous nucleic acid comprising at least one nucleic acid sequence operably linked to a promoter and encoding at least one viral protein, and controlling expression of the at least one viral protein using light. For example, in some embodiments, the at least one viral protein can be produced by the methods and compositions described herein by providing (or removing) light at a first wavelength or a wavelength range. In some embodiments, the methods and compositions described herein comprises introducing or providing at least one nucleic acid sequence encoding at least one light-controllable transcriptional regulator described herein, that interacts (directly or indirectly) with the promoter (e.g., an inducible promoter) described herein.

[0034] Further provided herein is the cell described in the methods as described herein or comprising the compositions as described herein. In some embodiments, the cell comprises at least one exogenous nucleic acid comprising at least one nucleic acid sequence operably linked to a promoter and encoding at least one viral protein. In some embodiments, the cell further comprises at least one light-controllable transcriptional regulator or at least one nucleic acid sequence encoding thereof, wherein the at least one light-controllable transcriptional regulator is activatable, upon modulated exposure to a light, to induce expression of the at least one viral protein under the control of the promote.

[0035] In some embodiments, the methods and composition described herein comprises generating a packaging cell line comprising the exogenous nucleic acid described herein. In some embodiments, the methods and composition described herein comprises generating a producer cell line comprising the exogenous nucleic acid described herein. In some embodiments, the methods and composition described herein further comprises a bioreactor comprising a plurality of cells described herein.WSGR Docket No. 60885-743.601

[0036] In some embodiments, the controlling the expression of the at least one viral protein using light comprises controlling the expression of the at least one viral protein via a temporary modulated exposure of the cell to the light. In some embodiments, a baseline culture condition of the cell is having the cell exposed to the light, and the temporary modulated exposure is provided by supplying the light to the cell (“ON”) and subsequently removing the light from the cell (“OFF”). Thus, the temporary modulated exposure to the light may be a temporary exposure to the light. Alternatively, the baseline culture condition of the cell is having the cell exposed to the light, and the temporary modulated exposure is provided by removing the light from the cell (“OFF”) and subsequently supplying the light to the cell (“ON”). Thus, the temporary modulated exposure to the light may be a temporary removal of the light.

[0037] In some embodiments, the temporary modulated exposure effects transient expression of the at least one viral protein in the cell. For example, the transient expression can be characterized by an on / off pattern of expression, such that the expression of the at least one viral protein is initiated and subsequently terminated within a defined time period. The transient expression can also be characterized by a variable expression profile comprising an initial increased rate of expression followed by a decreased rate of expression over time. In some embodiments, transient expression can further comprise, in a non-limiting manner, pulse-like expression in discrete bursts, gradual ramp-up and shut-down of expression, stepwise expression in multiple stages, oscillatory expression with fluctuations between higher and lower levels, or time-limited expression that persists for a predetermined duration.

[0038] In some embodiments, the cycle between ON and OFF states of the light (e.g., for the temporary exposure to the light or temporary removal of the light) for the temporary light exposure can be performed once or can be performed a plurality of times, e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times; or at most 50 times, at most 40 times, at most 30 times, at most 20 times, at most 10 times, at most 5 times, at most 4 times, at most 3 times, or at most 2 times. The ON state and the OFF state may have the same or different duration of time. For example, the ON state may be longer in time than the OFF state. Alternatively, the OFF state maybe longer in time than the ON state.

[0039] In some embodiments, a duration of the temporary modulated exposure is between about 0.1 hours and about 360 hours. In some embodiments, the duration of the temporary modulated exposure is between about 0.1 hours and about 0.5 hours, about 0.1 hours and about 1 hour, about 0.1 hours and about 2 hours, about 0.1 hours and about 5 hours, about 0.1 hours and about 10 hours, about 0.1 hours and about 15 hours, about 0.1 hours and about 20 hours, about 0.1 hours and about 25 hours, about 0.1 hours and about 30 hours, about 0.1 hours and about 35WSGR Docket No. 60885-743.601hours, about 0.1 hours and about 40 hours, about 0.1 hours and about 50 hours, about 0.1 hours and about 60 hours, about 0.1 hours and about 70 hours, about 0.1 hours and about 80 hours, about 0.1 hours and about 90 hours, about 0.1 hours and about 100 hours, about 0.1 hours and about 120 hours, about 0.1 hours and about 150 hours, about 0.1 hours and about 180 hours, about 0.1 hours and about 200 hours, about 0.1 hours and about 220 hours, about 0.1 hours and about 250 hours, about 0.1 hours and about 300 hours, about 0.5 hours and about 48 hours, about 0.5 hours and about 60 hours, about 0.5 hours and about 100 hours, about 0.5 hours and about 200 hours, about 0.5 hours and about 360 hours, about 3 hours and about 48 hours, about 3 hours and about 60 hours, about 3 hours and about 120 hours, about 3 hours and about 240 hours, about 3 hours and about 360 hours, about 8 hours and about 48 hours, about 8 hours and about 60 hours, about 8 hours and about 120 hours, about 8 hours and about 240 hours, about 8 hours and about 360 hours, about 12 hours and about 48 hours, about 12 hours and about 60 hours, about 12 hours and about 120 hours, about 12 hours and about 240 hours, about 12 hours and about 360 hours, about 24 hours and about 48 hours, about 24 hours and about 60 hours, about 24 hours and about 120 hours, about 24 hours and about 240 hours, about 24 hours and about 360 hours, about 48 hours and about 60 hours, about 48 hours and about 120 hours, about 48 hours and about 240 hours, about 48 hours and about 360 hours, about 60 hours and about 120 hours, about 60 hours and about 240 hours, about 60 hours and about 360 hours, about 120 hours and about 240 hours, about 120 hours and about 360 hours, about 240 hours and about 300 hours, about 240 hours and about 360 hours, or about 300 hours and about 360 hours. In some embodiments, a duration of the temporary modulated exposure is between about 0.5 hours and 60 hours. In some embodiments, the duration of the temporary modulated exposure is between about 6 hours and 60 hours. In some embodiments, the duration of the temporary modulated exposure is between about 10 hours and 60 hours. In some embodiments, the duration of the temporary modulated exposure is between about 24 hours and 60 hours.

[0040] In some embodiments, the duration of the temporary modulated exposure is at least about 0.1 hours, at least about 0.2 hours, at least about 0.5 hours, at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, at least about 48 hours, at least about 54 hours, at least about 60 hours, at least about 66 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, at least about 168 hours, at least about 192 hours, at least about 216 hours, at least about 240 hours, at least about 264 hours, at least about 288 hours, at least about 312 hours, at least about 336 hours, or at least about 360 hours. In some embodiments, the duration of the temporary modulated exposure is at most about 360 hours, at most about 336 hours, at most about 312 hours,WSGR Docket No. 60885-743.601at most about 288 hours, at most about 264 hours, at most about 240 hours, at most about 216 hours, at most about 192 hours, at most about 168 hours, at most about 144 hours, at most about 120 hours, at most about 96 hours, at most about 84 hours, at most about 72 hours, at most about 66 hours, at most about 60 hours, at most about 54 hours, at most about 48 hours, at most about 42 hours, at most about 36 hours, at most about 30 hours, at most about 24 hours, at most about 18 hours, at most about 12 hours, at most about 6 hours, at most about 2 hours, at most about 1 hour, at most about 0.5 hours, at most about 0.2 hours, or at most about 0.1 hours.

[0041] In some embodiments, the temporary modulated exposure comprises a continuous temporary modulated exposure. Continuous temporary modulated exposure can comprise (i) uninterrupted or substantially uninterrupted application of the light for a defined period of time (e.g., for temporary exposure to the light) or (ii) uninterrupted or substantially uninterrupted removal of the light from the cell for a defined period of time (e.g., for temporary removal of the light).

[0042] In some embodiments, the temporary exposure comprises a pulsed exposure, in which light is applied intermittently. The pulsed exposure can comprise regular or irregular intervals of illumination separated by periods of no illumination. For example, the pulsed exposure can occur at microsecond-scale, millisecond-scale, second-scale intervals, or minute-scale intervals, and can be applied at frequencies ranging from about 0.001 Hz to about 10 kHz, about 0.001 Hz to about 10 kHz, about 0.01 Hz to about 10 kHz, about 0.1 Hz to about 10 kHz, about 0.1 Hz to about 1 kHz, about 0.1 Hz to about 100 Hz, about 0.1 Hz to about 10 Hz, or about 0.1 Hz to about 1 Hz. In some embodiments, the pulsed exposure can comprise ON periods and OFF periods. For example, the ON period can be at least about 1 second, at least about 10 seconds, at least about 30 second, at least about 60 seconds, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, at least about 120 minutes, at least about 240 minutes, or at least about 360 minutes. In some embodiments, the OFF period can be at least about 1 second, at least about 10 seconds, at least about 30 second, at least about 60 seconds, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, at least about 120 minutes, at least about 240 minutes, or at least about 360 minutes. In some embodiments, the ON / OFF timing comprises asymmetric cycles or duty cycles. In some embodiments, the duty cycles rangeWSGR Docket No. 60885-743.601from about 1% to about 99%. In some embodiments, the temporary exposure comprises sinusoidal modulation, square-wave modulation, ramped intensity profiles, or combinations thereof.

[0043] In some embodiments, even when the light is provided by an optical source (e.g., LED lights) in a continuous manner, the exposure of the cells to the light may be interrupted by virtue of the cell culture set up. For example, the cells may be cultured in a bioreactor, whereby the optical source is directing the light towards the bioreactor, and the cells may be moving within the bioreactor (e.g., due to fluid flow within the bioreactor) relative to the optical pathway of the light from the optical source to the bioreactor, thereby having disrupted exposure to the light when the cells is moving away or outside of the optical pathway. In some cases, the term “pulsed exposure” of the light as provided herein may not refer to such disrupted exposure to the light by virtue of the movement of the cell. Instead, the term “pulsed exposure” of the light may refer to the function of the optical source in providing such light in such pulsed pattern. In some cases, the “continuous temporary exposure” of the light as provided herein may refer to the time that the optical source is providing the light to the cells (or to a cell culture device such as a bioreactor) regardless of the movement of the cells relative to the optical path of the light.

[0044] In some embodiments, the method further comprises culturing the cell for a culture period to produce a virus particle. The virus particle may comprise the at least one viral protein (e.g., when expression of a Cap protein is under control of the light). Alternatively or in addition to, production of the virus particle may be mediated by the at least one viral protein while the at least one viral protein does not become part of the viral particle (e.g., when expression of a helper protein and / or a Rep protein is under control the light). In some embodiments, the culture period is between about 0.1 hours and about 360 hours. In some embodiments, the culture period is between about 0.1 hours and about 0.5 hours, about 0.1 hours and about 1 hour, about 0.1 hours and about 2 hours, about 0.1 hours and about 5 hours, about 0.1 hours and about 10 hours, about 0.1 hours and about 15 hours, about 0.1 hours and about 20 hours, about 0.1 hours and about 25 hours, about 0.1 hours and about 30 hours, about 0.1 hours and about 35 hours, about 0.1 hours and about 40 hours, about 0.1 hours and about 50 hours, about 0.1 hours and about 60 hours, about 0.1 hours and about 70 hours, about 0.1 hours and about 80 hours, about 0.1 hours and about 90 hours, about 0.1 hours and about 100 hours, about 0.1 hours and about 120 hours, about 0.1 hours and about 150 hours, about 0.1 hours and about 180 hours, about 0.1 hours and about 200 hours, about 0.1 hours and about 220 hours, about 0.1 hours and about 250 hours, about 0.1 hours and about 300 hours, about 0.5 hours and about 48 hours, about 0.5 hours and about 60 hours, about 0.5 hours and about 100 hours, about 0.5 hours and about 200 hours, about 0.5 hours and about 360 hours, about 3 hours and about 48 hours, about 3 hours and about 60 hours, about 3 hours and about 120 hours, about 3 hours and about 240 hours, about 3 hours and about 360 hours, about 8WSGR Docket No. 60885-743.601hours and about 48 hours, about 8 hours and about 60 hours, about 8 hours and about 120 hours, about 8 hours and about 240 hours, about 8 hours and about 360 hours, about 12 hours and about 48 hours, about 12 hours and about 60 hours, about 12 hours and about 120 hours, about 12 hours and about 240 hours, about 12 hours and about 360 hours, about 24 hours and about 48 hours, about 24 hours and about 60 hours, about 24 hours and about 120 hours, about 24 hours and about 240 hours, about 24 hours and about 360 hours, about 48 hours and about 60 hours, about 48 hours and about 120 hours, about 48 hours and about 240 hours, about 48 hours and about 360 hours, about 60 hours and about 120 hours, about 60 hours and about 240 hours, about 60 hours and about 360 hours, about 120 hours and about 240 hours, about 120 hours and about 360 hours, about 240 hours and about 300 hours, about 240 hours and about 360 hours, or about 300 hours and about 360 hours. In some embodiments, the culture period is between about 0.5 hours and 60 hours. In some embodiments, the culture period is between about 6 hours and 60 hours. In some embodiments, the culture period is between about 10 hours and 60 hours. In some embodiments, the culture period is between about 24 hours and 60 hours.

[0045] In some embodiments, the culture period may begin upon introducing at least one nucleic acid sequence encoding at least one viral protein to the cell (e.g., via transfection or transduction). Alternatively, the culture period may begin upon introducing genes encoding all necessary viral proteins to the cell. For example, the culture period may begin upon introducing both (i) one or more viral proteins having expression inducible by light and (ii) the rest of the necessary viral protein or proteins having expression inducible by an endogenous promoter and / or a viral promoter.

[0046] In some embodiments, the culture period is at least about 0.1 hours, at least about 0.2 hours, at least about 0.5 hours, at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, at least about 48 hours, at least about 54 hours, at least about 60 hours, at least about 66 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, at least about 168 hours, at least about 240 hours, at least about 300 hours, or at least about 360 hours. In some embodiments, the culture period is at most about 360 hours, at most about 300 hours, at most about 240 hours, at most about 168 hours, at most about 144 hours, at most about 120 hours, at most about 96 hours, at most about 84 hours, at most about 72 hours, at most about 66 hours, at most about 60 hours, at most about 54 hours, at most about 48 hours, at most about 42 hours, at most about 36 hours, at most about 30 hours, at most about 24 hours, at most about 18 hours, at most about 12 hours, at most about 6 hours, at most about 2 hours, at most about 1 hour, at most about 0.5 hours, at most about 0.2 hours, or at most about 0.1 hours.WSGR Docket No. 60885-743.601

[0047] In some embodiments, the temporary modulated exposure is initiated during the initial 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the culture period. In some embodiments, the temporary modulated exposure is initiated during the initial 50% of the culture period. In some embodiments, the temporary modulated exposure is initiated during the initial 30% of the culture period. In some embodiments, the temporary modulated exposure is initiated within the initial 0.1 hours, 0.2 hours, 0.3 hours, 0.4 hours, 0.5 hours, 0.6 hours, 0.7 hours, 0.8 hours, 0.9 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, 168 hours, or 180 hours of the culture period. In some embodiments, the temporary modulated exposure is initiated within the initial 36 hours of the culture period. In some embodiments, the temporary modulated exposure is initiated within the initial 24 hours of the culture period.

[0048] In some embodiments, a duration of the temporary modulated exposure is shorter than the culture period by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, a duration of the temporary modulated exposure is shorter than the culture period by at most about 99%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, or at most about 5%. In some embodiments, a duration of the temporary modulated exposure is shorter than the culture period by at least about 10%, at least about 20%, or at least about 30%. In some embodiments, a duration of the temporary modulated exposure is shorter than the culture period by at most about 50%, at most about 40%, or at most about 30%.

[0049] In some embodiments, the temporary modulated exposure of the cell to the light yields a production level of the virus particle that is greater than that produced via use of a comparable temporary modulated exposure to the light (e.g., substantially the same duration of time as the temporary modulated exposure) that is initiated at a later time point during the culture period than the temporary modulated exposure. In some embodiments, the production level of the virusWSGR Docket No. 60885-743.601particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, or at least about 5000%. In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at least about 10%, at least about 50%, at least about 100%, or at least about 200%.

[0050] In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at most about 5000%, at most about 4000%, at most about 3000%, at most about 2000%, at most about 1000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 350%, at most about 300%, at most about 250%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, or at most about 5%. In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at most about 4000%, at most about 2000%, at most about 1000%, or at most about 300%. In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11 -fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at leastWSGR Docket No. 60885-743.601about 21 -fold, at least about 22-fold, at least about 23 -fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 70-fold, or at least about 100-fold. In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at least about 1-fold, at least about 2-fold, at least about 3-fold, or at least about 5 -fold.

[0051] In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at most about 100-fold, at most about 70-fold, at most about 50-fold, at most about 40-fold, at most about 35-fold, at most about 30-fold, at most about 29-fold, at most about 28-fold, at most about 27-fold, at most about 26-fold, at most about 25-fold, at most about 24-fold, at most about 23-fold, at most about 22-fold, at most about 21 -fold, at most about 20-fold, at most about 19-fold, at most about 18-fold, at most about 17-fold, at most about 16-fold, at most about 15-fold, at most about 14-fold, at most about 13 -fold, at most about 12-fold, at most about 11 -fold, at most about 10-fold, at most about 9-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5 -fold, at most about 4-fold, at most about 3 -fold, at most about 2-fold, at most about 1-fold, or at most about 0.5-fold. In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at most about 40-fold, at most about 20-fold, or at most about 10-fold.

[0052] In some embodiments, the temporary modulated exposure of the cell to the light yields a production level of the virus particle that is greater than that produced by via use of a longer exposure to the light. In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, or at least about 5000%. In some embodiments, the production level of the virus particle via the temporaryWSGR Docket No. 60885-743.601modulated exposure is greater than that via the longer exposure by at least about 10%, at least about 50%, at least about 100%, or at least about 200%.

[0053] In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at most about 5000%, at most about 4000%, at most about 3000%, at most about 2000%, at most about 1000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 350%, at most about 300%, at most about 250%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, or at most about 5%. In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at most about 1000%, at most about 500%, or at most about 200%.

[0054] In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at least about 0.1 -fold, by at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13 -fold, at least about 14-fold, at least about 15 -fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21 -fold, at least about 22-fold, at least about 23 -fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 70-fold, or at least about 100-fold. In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at least about 0.5-fold, at least about 1-fold, or at least about 2-fold.

[0055] In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at most about 100-fold, at most about 70-fold, at most about 50-fold, at most about 40-fold, at most about 35-fold, at most about 30-fold, at most about 29-fold, at most about 28-fold, at most about 27-fold, at most about 26-fold, at most about 25 -fold, at most about 24-fold, at most about 23 -fold, at most about 22-fold, at most about 21 -fold, at most about 20-fold, at most about 19-fold, at most about 18-fold, at mostWSGR Docket No. 60885-743.601about 17-fold, at most about 16-fold, at most about 15-fold, at most about 14-fold, at most about 13 -fold, at most about 12-fold, at most about 11 -fold, at most about 10-fold, at most about 9-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, at most about 1-fold, at most about 0.5-fold, or at most about O.l-fold. In some embodiments, the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at most about 10-fold, at most about 5-fold, or at most about 3-fold.

[0056] In some embodiments, a nucleic acid encoding any one of the viral proteins described herein can be engineered or modified such that it lacks a functional internal promoter and / or a functional start codon. In some embodiments, the modification can prevent unintended transcriptional or translational initiation from cryptic internal elements. In some embodiments, such modification prevents expression of a smaller viral protein from a gene encoding both the smaller viral protein and a larger viral protein, such that only a larger viral protein is expressed under the control of an upstream regulatory element. In some embodiments, the viral protein comprises viral replication protein. In some embodiments, the smaller viral protein comprises Rep52 or Rep40, which, before such modification, can be expressed from the pl9 promoter and translated from the internal start codons located within the coding sequence of larger replication proteins such as Rep68 or Rep78. In some embodiments, the larger viral protein (e.g., Rep68 or Rep78) is expressed under the control of a heterologous promoter or its native p5 promoter. In one example, the large Rep protein Rep68 comprises a methionine-to-glycine substitution at residue 225 (M225G), which disrupts the internal start codon responsible for initiating translation of Rep40 within the Rep68 coding sequence.

[0057] In some embodiments, the at least one nucleic acid sequence does not comprise a functional internal promoter associated with expression of the at least one viral protein in a native viral context, e.g., expression of the at least one viral protein is instead under the control of a heterologous promoter such as an optogenetic promoter (e.g., activation of the promoter is inducible by light via, for example, a light controllable transcriptional regulator described herein) or a non-optogenetic promoter (e.g., a constitutive promoter). In some embodiments, the functional internal promoters comprise promoters that regulate transcription of viral replication proteins, structural proteins, and / or accessory proteins in the native viral context. In some embodiments, the native viral context is in the context of lentivirus. In some embodiments, the internal promoters comprise the long terminal repeat (LTR) promoter that drives transcription of gag, pol, and env genes encoding structural and enzymatic proteins. In some embodiments, the native viral context is in the context of adeno-associated virus (AAV). In some embodiments, the internal promoters comprise the endogenous p5 promoter that drives transcription of large RepWSGR Docket No. 60885-743.601proteins (e.g., Rep78 and Rep68), the pl9 promoter that drives transcription of smaller Rep proteins (e.g., Rep52 and Rep40), or the p40 promoter that drives transcription of capsid proteins (e.g., VP1, VP2, and VP3). In some embodiments, the native viral context comprises helper virus functions required for AAV replication. In some embodiments, the functional internal promoters comprise promoters for adenovirus or herpesvirus helper genes. In some embodiments, the functional internal promoters comprise the adenoviral E1A promoter (e.g., for driving transcription of El A gene), the adenoviral E2A promoter (e.g., for driving transcription of E2A gene), the adenoviral E4 promoter (e.g., for driving transcription of E4orf6 gene), or the adenoviral major late promoter (MLP) (e.g., for driving transcription of the L4 gene).

[0058] In some embodiments, the at least one nucleic acid sequence does not comprise a gene encoding the functional internal promoter. In some embodiments, the internal promoter can be rendered non -functional by one or more modifications. In some embodiments, the modifications comprise deletion of the promoter sequence, mutation of critical regulatory elements (e.g., TATA box, transcription factor binding sites, insertion of transcriptional terminators or insulator sequences upstream of the promoter, inversion of the promoter sequence, replacement of the promoter with a non-functional sequence, or any combination thereof. In some embodiments, the internal promoter may be altered, mutated, or otherwise modified to abolish or substantially reduce its activity, such that it is incapable of driving expression of the viral protein. In some embodiments, the internal promoter is inactivated by transcriptional silencing. Silencing can comprise epigenetic modification (e.g., methylation of CpG islands, histone modification), recruitment of repressor proteins, or use of RNA interference targeting promoter-associated transcripts. In some embodiments, silencing is achieved without altering the coding sequence of the viral protein, thereby allowing expression under an alternative heterologous promoter (e.g., the standalone Rep40 not having its native viral promoter but instead under the control of an inducible promoter as shown in FIG. 19). In some embodiments, the silencing mechanism disrupts promoter activity without deleting or mutating the promoter sequence. In some embodiments, the silencing mechanism introduces insulator elements, chromatin remodeling factors, or other regulatory elements that block transcription initiation at the internal promoter.

[0059] In some embodiments, the cell comprises an additional nucleic acid sequence encoding an additional promoter and an additional viral protein, wherein the additional promoter is not inducible by the light (e.g., inducible by a different light, inducible by a different non-light system such as a small molecule inducible system, or a non-inducible promoter such as a constitutive promoter), and wherein the at least one viral protein and the additional viral protein are different. In some embodiments, the at least one exogenous nucleic acid and the additional exogenous nucleic acid are different nucleic acid molecules. In some embodiments, the at leastWSGR Docket No. 60885-743.601one exogenous nucleic acid and the additional exogenous nucleic acid are within a single nucleic acid molecule. In some embodiments, the additional promoter comprises a viral promoter (e.g., a functional viral promoter). In some embodiments, the additional promoter comprises a promoter that is not a native promoter to the additional viral protein under the control of the additional promoter. For example, such a promoter may be a constitutive promoter or an inducible promoter that is inducible by a trigger or mechanism other than the light (e.g., a different light, a small molecule, etc.).

[0060] In some embodiments, (i) the at least one viral protein comprises a helper protein and (ii) the additional viral protein comprises a replication protein, a capsid protein, or both. In some embodiments, (i) the at least one viral protein comprises a replication protein and (ii) the additional viral protein comprises a helper protein, a capsid protein, or both. In some embodiments, (i) the at least one viral protein comprises a capsid protein and (ii) the additional viral protein comprises a helper protein, a replication protein, or both.

[0061] In some embodiments, the at least one viral protein, the additional viral protein, and the viral promoter are derived from the same type of virus. In some embodiments, the same type of virus comprises an adeno-associated virus (AAV). In some embodiments, the viral proteins are derived from different viruses. In some cases, an AAV replication protein and capsid protein can be combined with an adenovirus helper protein. In some cases, the Rep proteins are derived from a first AAV type (e.g., AAV-2) and the capsid proteins are from a second AAV type (e.g., another AAV serotype). For example, AAV therapeutics may differ in their capsid proteins but may be produced with the Rep proteins from AAV-2. Thus, a packaging cell as provided herein may be universal for different Cap proteins (e.g., the packing cell may have expression systems for helper genes and Rep protein genes, such that different Cap genes of interest can be introduced for different applications).

[0062] In some embodiments, the helper protein as provided herein is derived from a helper virus. In some embodiments, the helper virus is adenovirus or herpesvirus. In some embodiments, the helper protein comprises an El A, E2A, E4 (e.g., E4orf6, E4orf6 / 7, or both), or L4. In some embodiments, the helper protein (e.g., an adenovirus helper protein) comprises one or more members selected from the group consisting of E2A, E4, and L4. The helper protein can comprise (i) at least E2A, (ii) at least E2A and E4orf6, or (ii) at least E2A, E4, and L4. In some embodiments, the viral promoter (e.g., the functional viral promoter, the non -functional or deactivated viral promoter as provided herein) comprises the p5 promoter that drives transcription of large Rep proteins (e.g., Rep78 and / or Rep68), the pl9 promoter that drives transcription of smaller Rep proteins (e.g., Rep52 and / or Rep40), or the p40 promoter that drives transcription of capsid proteins (e.g., VP1, VP2, and / or VP3).WSGR Docket No. 60885-743.601

[0063] In some embodiments, the at least one viral protein or the additional viral protein comprises a replication protein, and such replication protein may comprise at least a pair of a large Rep protein and a small Rep protein. For example, expression of the large Rep protein and the small Rep protein may be controlled by the light (e.g., under the control of the light controllable transcriptional regulator). The pair may comprise Rep68 and Rep40. Alternatively or in addition to, the pair may comprise Rep78 and Rep 52. Such pair may be encoded by a same (or single) nucleic acid molecule or each encoded by a different nucleic acid molecule.

[0064] In some embodiments, at least a portion of a gene encoding a viral protein as provided herein (e.g., the at least one nucleic acid sequence encoding the at least one viral protein, the additional nucleic acid sequence encoding the additional viral protein, etc.) may be codon optimized. For example, the gene encoding the viral protein (e.g., Rep proteins, such as Rep40, Rep52, Rep68, Rep78) may be under the control of a light inducible promoter, and such gene may be codon optimized. The term “codon optimization” as used herein generally refers to improving and / or maximizing the protein expression in a living organism or cell by increasing the translational efficiency of gene of interest by altering the nucleotides used to encode the same amino acid residue(s) (e.g., transforming / replacing DNA sequence of nucleotides of one species into DNA sequence of nucleotides of another species. Codon optimization involves replacing wild type DNA sequences and rare codons by more highly expressed species sequences and frequently occurring codons without changing the protein. For example, codon optimization of viral genes (e.g., Rep or Cap proteins) can be utilized for production of the viral particles in human cell production system (e.g., HEK293F cells). Alternatively or in addition to, codon optimization may be used to ablate internal functional elements (e.g., promoters) and maintaining high expression (e.g., codon optimization 2 in FIG. 19, where the codon optimization yielded (i) maximized distance to the original sequence and (ii) maximized codon usage). Codon optimization may maximize expression of a gene of interest and / or remove internal functional / regulatory elements.

[0065] In some embodiments, the at least one viral protein comprises at least a helper protein under control of at least one optogenetic promoter, and the cell further comprises at least a replication protein, at least a capsid protein, and at least a gene of interest (GO I) under control of at least one non-optogenetic promoter. In some embodiments, the at least one viral protein comprises at least a helper protein and at least a replication protein under control of at least one optogenetic promoter, and the cell further comprises at least a capsid protein and at least a gene of interest (GO I) under control of at least one non-optogenetic promoter. In some embodiments, the at least one viral protein comprises at least a helper protein and at least a capsid protein under control of at least one optogenetic promoter, and the cell further comprises at least a replication protein and at least a gene of interest (GOI) under control of at least one non-optogenetic promoter.WSGR Docket No. 60885-743.601In some embodiments, the at least one viral protein comprises at least a helper protein and at least a gene of interest (GO I) protein under control of at least one optogenetic promoter, and the cell further comprises at least a replication protein and at least a capsid protein under control of at least one non-optogenetic promoter. In some embodiments, the at least one viral protein comprises at least a replication protein under control of at least one optogenetic promoter, and the cell further comprises at least a helper protein, at least a capsid protein, and at least a gene of interest (GOI) under control of at least one non-optogenetic promoter. In some embodiments, the at least one viral protein comprises at least a replication protein and at least a capsid protein under control of at least one optogenetic promoter, and the cell further comprises at least a helper protein and at least a gene of interest (GOI) under control of at least one non-optogenetic promoter. In some embodiments, the at least one viral protein comprises at least a replication protein and at least a gene of interest (GOI) under control of at least one optogenetic promoter, and the cell further comprises at least a helper protein and at least a capsid protein under control of at least one non-optogenetic promoter. In some embodiments, the at least one viral protein comprises at least a capsid protein under control of at least one optogenetic promoter, and the cell further comprises at least a helper protein, at least a replication protein, and at least a gene of interest (GOI) under control of at least one non-optogenetic promoter. In some embodiments, the at least one viral protein comprises at least a capsid protein and at least a gene of interest (GOI) under control of at least one optogenetic promoter, and the cell further comprises at least a helper protein and at least a replication protein under control of at least one non-optogenetic promoter. In some embodiments, the at least one viral protein comprises at least a capsid protein, at least a helper protein, and at least a replication protein under control of at least one optogenetic promoter, and the cell further comprises at least a gene of interest (GOI) under control of at least one non-optogenetic promoter.

[0066] In some embodiments, the at least one helper protein, at least one replication protein, at least one capsid protein, or at least one gene of interest (GOI) is operably linked to one or more promoters. In some embodiments, the at least one helper protein, at least one replication protein, at least one capsid protein, or at least one gene of interest (GOI) is expressed under control of at least one optogenetic promoter. In some embodiments, at least one of the remaining components is not expressed under control of an optogenetic promoter. In some embodiments, at least one of the remaining components is expressed under control of at least one non-optogenetic promoter. In some embodiments, the non-optogenetic promoter is constitutive or tissue-specific promoter. In some embodiments, the non-optogenetic promoter comprises CMV, EFla, CAG, UbC, PGK, or variants thereof. In some embodiments, the non-optogenetic promoter comprises a viral promoter. In some embodiments, the non-optogenetic promoter comprises p5, pl 9, p40, orWSGR Docket No. 60885-743.601variants thereof. In some embodiments, multiple optogenetic promoters and non-optogenetic promoters are used.

[0067] In some embodiments, the at least one viral protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen or at least twenty viral proteins.

[0068] In some embodiments, the methods and compositions described herein comprise providing or obtaining a cell comprising at least one exogenous nucleic acid. In some embodiments, the at least one exogenous nucleic acid comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen or at least twenty exogenous nucleic acids.

[0069] In some embodiments, the at least one exogenous nucleic acid comprises a first nucleic acid sequence described herein. In some embodiments, the at least one exogenous nucleic acid comprises the first nucleic acid sequence encoding at least one viral protein described herein. In some embodiments, the at least one exogenous nucleic acid comprises at least one promoter described herein. In some embodiments, the at least one promoter comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen or at least twenty promoters.

[0070] In some embodiments, the at least one exogenous nucleic acid comprises at least one promoter operably linked to the at least one viral protein described herein. In some embodiments, the at least one exogenous nucleic acid comprises a second nucleic acid sequence described herein. In some embodiments, the at least one exogenous nucleic acid comprises a second nucleic acid sequence encoding a light controllable transcriptional regulator described herein. In some embodiments, the methods and compositions described herein comprises controlling the expression of the at least one viral protein using light.

[0071] In some embodiments, the at least one exogenous nucleic acid described herein comprises DNA. In some embodiments, the DNA comprises single-stranded and double-stranded DNA. In some embodiments, the DNA comprises modified deoxyribonucleotides.

[0072] In some embodiments, the at least one exogenous nucleic acid described herein comprises an RNA molecule. In some embodiments, the RNA comprises single-stranded and double-stranded DNA. In some embodiments, the RNA comprises modified ribonucleotides.

[0073] In some embodiments, the size of the at least one exogenous nucleic acid can be up to about 0.1 kilobases to about 1000 kilobases. In some embodiments, the size of the at least oneWSGR Docket No. 60885-743.601exogenous nucleic acid can be up to about 0.1 kilobases to about 1,000 kilobases. In some embodiments, the size of the at least one exogenous nucleic acid can be up to about 0.1 kilobases to about 1 kilobases, about 0.1 kilobases to about 30 kilobases, about 0.1 kilobases to about 40 kilobases, about 0.1 kilobases to about 60 kilobases, about 0.1 kilobases to about 80 kilobases, about 0.1 kilobases to about 100 kilobases, about 0.1 kilobases to about 200 kilobases, about 0.1 kilobases to about 400 kilobases, about 0.1 kilobases to about 600 kilobases, about 0.1 kilobases to about 800 kilobases, about 0.1 kilobases to about 1,000 kilobases, about 1 kilobases to about 30 kilobases, about 1 kilobases to about 40 kilobases, about 1 kilobases to about 60 kilobases, about 1 kilobases to about 80 kilobases, about 1 kilobases to about 100 kilobases, about 1 kilobases to about 200 kilobases, about 1 kilobases to about 400 kilobases, about 1 kilobases to about 600 kilobases, about 1 kilobases to about 800 kilobases, about 1 kilobases to about 1,000 kilobases, about 30 kilobases to about 40 kilobases, about 30 kilobases to about 60 kilobases, about 30 kilobases to about 80 kilobases, about 30 kilobases to about 100 kilobases, about 30 kilobases to about 200 kilobases, about 30 kilobases to about 400 kilobases, about 30 kilobases to about 600 kilobases, about 30 kilobases to about 800 kilobases, about 30 kilobases to about 1,000 kilobases, about 40 kilobases to about 60 kilobases, about 40 kilobases to about 80 kilobases, about 40 kilobases to about 100 kilobases, about 40 kilobases to about 200 kilobases, about 40 kilobases to about 400 kilobases, about 40 kilobases to about 600 kilobases, about 40 kilobases to about 800 kilobases, about 40 kilobases to about 1,000 kilobases, about 60 kilobases to about 80 kilobases, about 60 kilobases to about 100 kilobases, about 60 kilobases to about 200 kilobases, about 60 kilobases to about 400 kilobases, about 60 kilobases to about 600 kilobases, about 60 kilobases to about 800 kilobases, about 60 kilobases to about 1,000 kilobases, about 80 kilobases to about 100 kilobases, about 80 kilobases to about 200 kilobases, about 80 kilobases to about 400 kilobases, about 80 kilobases to about 600 kilobases, about 80 kilobases to about 800 kilobases, about 80 kilobases to about 1,000 kilobases, about 100 kilobases to about 200 kilobases, about 100 kilobases to about 400 kilobases, about 100 kilobases to about 600 kilobases, about 100 kilobases to about 800 kilobases, about 100 kilobases to about 1,000 kilobases, about 200 kilobases to about 400 kilobases, about 200 kilobases to about 600 kilobases, about 200 kilobases to about 800 kilobases, about 200 kilobases to about 1,000 kilobases, about 400 kilobases to about 600 kilobases, about 400 kilobases to about 800 kilobases, about 400 kilobases to about 1,000 kilobases, about 600 kilobases to about 800 kilobases, about 600 kilobases to about 1,000 kilobases, or about 800 kilobases to about 1,000 kilobases. In some embodiments, the size of the at least one exogenous nucleic acid can be up to about 0.1 kilobases, about 1 kilobases, about 30 kilobases, about 40 kilobases, about 60 kilobases, about 80 kilobases, about 100 kilobases, about 200 kilobases, about 400 kilobases, about 600 kilobases, about 800 kilobases, or about 1,000WSGR Docket No. 60885-743.601kilobases. In some embodiments, the size of the at least one exogenous nucleic acid can be up to at least about 0.1 kilobases, about 1 kilobases, about 30 kilobases, about 40 kilobases, about 60 kilobases, about 80 kilobases, about 100 kilobases, about 200 kilobases, about 400 kilobases, about 600 kilobases, or about 800 kilobases. In some embodiments, the size of the at least one exogenous nucleic acid can be up to at most about 1 kilobases, about 30 kilobases, about 40 kilobases, about 60 kilobases, about 80 kilobases, about 100 kilobases, about 200 kilobases, about 400 kilobases, about 600 kilobases, about 800 kilobases, or about 1,000 kilobases.

[0074] In some embodiments, the size of the at least one exogenous nucleic acid can be up to about 0.1 kilobases to about 50 kilobases. In some embodiments, the size of the at least one exogenous nucleic acid can be up to about 0.1 kilobases to about 1 kilobase, about 0.1 kilobases to about 5 kilobases, about 0.1 kilobases to about 10 kilobases, about 0.1 kilobases to about 15 kilobases, about 0.1 kilobases to about 20 kilobases, about 0.1 kilobases to about 25 kilobases, about 0.1 kilobases to about 30 kilobases, about 0.1 kilobases to about 35 kilobases, about 0.1 kilobases to about 40 kilobases, about 0.1 kilobases to about 45 kilobases, about 0.1 kilobases to about 50 kilobases, about 1 kilobase to about 5 kilobases, about 1 kilobase to about 10 kilobases, about 1 kilobase to about 15 kilobases, about 1 kilobase to about 20 kilobases, about 1 kilobase to about 25 kilobases, about 1 kilobase to about 30 kilobases, about 1 kilobase to about 35 kilobases, about 1 kilobase to about 40 kilobases, about 1 kilobase to about 45 kilobases, about 1 kilobase to about 50 kilobases, about 5 kilobases to about 10 kilobases, about 5 kilobases to about 15 kilobases, about 5 kilobases to about 20 kilobases, about 5 kilobases to about 25 kilobases, about 5 kilobases to about 30 kilobases, about 5 kilobases to about 35 kilobases, about 5 kilobases to about 40 kilobases, about 5 kilobases to about 45 kilobases, about 5 kilobases to about 50 kilobases, about 10 kilobases to about 15 kilobases, about 10 kilobases to about 20 kilobases, about 10 kilobases to about 25 kilobases, about 10 kilobases to about 30 kilobases, about 10 kilobases to about 35 kilobases, about 10 kilobases to about 40 kilobases, about 10 kilobases to about 45 kilobases, about 10 kilobases to about 50 kilobases, about 15 kilobases to about 20 kilobases, about 15 kilobases to about 25 kilobases, about 15 kilobases to about 30 kilobases, about 15 kilobases to about 35 kilobases, about 15 kilobases to about 40 kilobases, about 15 kilobases to about 45 kilobases, about 15 kilobases to about 50 kilobases, about 20 kilobases to about 25 kilobases, about 20 kilobases to about 30 kilobases, about 20 kilobases to about 35 kilobases, about 20 kilobases to about 40 kilobases, about 20 kilobases to about 45 kilobases, about 20 kilobases to about 50 kilobases, about 25 kilobases to about 30 kilobases, about 25 kilobases to about 35 kilobases, about 25 kilobases to about 40 kilobases, about 25 kilobases to about 45 kilobases, about 25 kilobases to about 50 kilobases, about 30 kilobases to about 35 kilobases, about 30 kilobases to about 40 kilobases, about 30 kilobases to about 45 kilobases,WSGR Docket No. 60885-743.601about 30 kilobases to about 50 kilobases, about 35 kilobases to about 40 kilobases, about 35 kilobases to about 45 kilobases, about 35 kilobases to about 50 kilobases, about 40 kilobases to about 45 kilobases, about 40 kilobases to about 50 kilobases, or about 45 kilobases to about 50 kilobases long.

[0075] In some embodiments, the size of the at least one exogenous nucleic acid can be up to about 0.1 kilobases, about 1 kilobase, about 5 kilobases, about 10 kilobases, about 15 kilobases, about 20 kilobases, about 25 kilobases, about 30 kilobases, about 35 kilobases, about 40 kilobases, about 45 kilobases, or about 50 kilobases. In some embodiments, the at least one exogenous nucleic acid is up to at least about 0.1 kilobases, about 1 kilobase, about 5 kilobases, about 10 kilobases, about 15 kilobases, about 20 kilobases, about 25 kilobases, about 30 kilobases, about 35 kilobases, about 40 kilobases, or about 45 kilobases. In some embodiments, the at least one exogenous nucleic acid is up to at most about 1 kilobase, about 5 kilobases, about 10 kilobases, about 15 kilobases, about 20 kilobases, about 25 kilobases, about 30 kilobases, about 35 kilobases, about 40 kilobases, about 45 kilobases, or about 50 kilobases long.

[0076] In some embodiments, providing or obtaining a cell described herein, such as a cell comprising at least one exogenous nucleic acid, comprises introducing at least one exogenous nucleic acid described herein into the cell. In some embodiments, the at least one exogenous nucleic acid is introduced into the cell using a vector described herein.

[0077] In some embodiments, a vector described herein comprises a DNA molecule. In some embodiments, the DNA molecule comprises a plasmid, a minicircle, an oligonucleotide, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a cosmid, a fosmid, a phagemid, a linear DNA fragment, or a synthetic DNA construct.

[0078] In some embodiments, the vector described herein comprises an RNA molecule. In some embodiments, the RNA molecule comprises a messenger RNA (mRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a long non-coding RNA (IncRNA), a circular RNA (circRNA), a guide RNA (gRNA), or an antisense RNA.

[0079] In some embodiments, the vector described herein can be introduced into the cell via transfection. In some embodiments, the transfection can comprise chemical transfection. In some embodiments, chemical transfection can comprise calcium phosphate transfection, DEAE-dextran transfection, or cationic lipid transfection. In some embodiments, the transfection can comprise physical transfection. In some embodiments physical transfection can comprise microinjection. In some embodiments, physical transfection can comprise electroporation.

[0080] In some embodiments, the vector can be introduced into the cell using a virus or viral vector. In some embodiments, the virus or viral vector comprises an Adeno-Associated Virus (AAV), aLentivirus, an Adenovirus, a Herpes Simplex Virus (HSV), a Retrovirus, aBaculovirus,WSGR Docket No. 60885-743.601a Poxvirus, a Sendai Virus, an Alphavirus, a Rabies Virus, a Cytomegalovirus (CMV), a Vesicular Stomatitis Virus (VSV), or a Foamy Virus.

[0081] In some embodiments, the at least one exogenous nucleic acid can comprise a first nucleic acid sequence encoding at least one viral protein. In some embodiments, the at least one viral protein can be codon-optimized. Different cells use certain codons more frequently to encode the same amino acid to match the amount of corresponding tRNAs in the cell. Codon-optimization can comprise modifying the codons within a gene to match the codons used most often to encode specific amino acids by the host cell that will express the gene. Codon-optimization ensures efficient ribosome recognition, minimizes translational pausing, and improves the overall efficiency of protein production in the host cell. Codon-optimization can further be used to eliminate the function of naturally occurring internal promoters such as the pl9 promoter.

[0082] In some embodiments, the at least one viral protein described herein comprises a lentiviral protein. In some embodiments, the lentiviral protein comprises a lentiviral Groupspecific Antigen (Gag) protein, lentiviral Polymerase (Pol) protein, a lentiviral Envelope (Env) protein, a lentiviral accessory protein, a lentiviral regulatory protein, any variant thereof or any combination thereof.

[0083] In some embodiments, the lentiviral Gag protein comprises a Matrix (MA) protein, a Capsid (CA) protein, aNucleocapsid (NC) protein, any variant thereof or any combination thereof.

[0084] In some embodiments, a protein or protein variant described herein comprises or consist of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to the wild-type proteins encoded on the Human Immunodeficiency Virus-1 (HIV-1) genome or genome variant described herein.

[0085] In some embodiments, the at least one viral protein described herein comprises an AAV protein. In some embodiments, the AAV protein can comprise a protein from an AAV1, an AAV2 , an AAV3 , an AAV4 , an AAV5 , an AAV6 , an AAV7 , an AAV8 , an AAV9 , an AAV10 , an AAV11 , an AAV12 , an AAV13 , a variant thereof, or any combination thereof.

[0086] In some embodiments, the variants of the AAV proteins described herein comprise or consist of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type AAV proteins or protein variants described herein.WSGR Docket No. 60885-743.601

[0087] In some embodiments, the AAV protein described herein comprises an AAV Replicase (Rep) protein. In some embodiments, the AAV Rep protein does not comprise a functional internal pl9 promoter. In some embodiments, AAV Rep protein described herein comprises an AAV Rep78 protein, an AAV Rep68, an AAV Rep52 or an AAV Rep 40 protein, a variant thereof, or any combination thereof.

[0088] In some embodiments, the variants of the AAV Rep78 protein described herein comprise or consist of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type AAV Rep78 proteins or protein variants (e.g., such as an AAV1 Rep78 protein, an AAV2 Rep78 protein, an AAV3 Rep78 protein, an AAV4 Rep78 protein, an AAV5 Rep78 protein, an AAV6 Rep78 protein, an AAV7 Rep78 protein, an AAV8 Rep78 protein, an AAV9 Rep78 protein, an AAV 10 Rep78 protein, an AAV11 Rep78 protein, an AAV 12 Rep78 protein, or an AAV 13 Rep78 protein) described herein.

[0089] In some embodiments, the variants of the AAV Rep68 protein described herein comprise or consist of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type AAV Rep68 proteins or protein variants (e.g., such as an AAV1 Rep68 protein, an AAV2 Rep68 protein, an AAV3 Rep68 protein, an AAV4 Rep68 protein, an AAV5 Rep68 protein, an AAV6 Rep68 protein, an AAV7 Rep68 protein, an AAV8 Rep68 protein, an AAV9 Rep68 protein, an AAV10 Rep68 protein, an AAV11 Rep68 protein, an AAV12 Rep68 protein, or an AAV13 Rep68 protein) described herein.

[0090] In some embodiments, the variants of the AAV Rep52 protein described herein comprise or consist of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type AAV Rep52 proteins or protein variants (e.g., such as an AAV1 Rep52 protein, an AAV2 Rep52 protein, an AAV3 Rep52 protein, an AAV4 Rep52 protein, an AAV5 Rep52 protein, an AAV6 Rep52 protein, an AAV7 Rep52 protein, an AAV8 Rep52 protein, an AAV9 Rep52 protein, an AAV10 Rep52 protein, an AAV11 Rep52 protein, an AAV12 Rep52 protein, or an AAV13 Rep52 protein) described herein.WSGR Docket No. 60885-743.601

[0091] In some embodiments, the variants of the AAV Rep40 protein described herein comprise or consist of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type AAV Rep40 proteins or protein variants (e.g., such as an AAV1 Rep40 protein, an AAV2 Rep40 protein, an AAV3 Rep40 protein, an AAV4 Rep40 protein, an AAV5 Rep40 protein, an AAV6 Rep40 protein, an AAV7 Rep40 protein, an AAV8 Rep40 protein, an AAV9 Rep40 protein, an AAV 10 Rep40 protein, an AAV11 Rep40 protein, an AAV 12 Rep40 protein, or an AAV 13 Rep40 protein) described herein.

[0092] In some embodiments, the AAV protein comprises an AAV Capsid protein. In some embodiments, the AAV Capsid protein comprises a VP1, a VP2 or a VP3 protein, any variant thereof, or any combination thereof.

[0093] In some embodiments, the variants of the AAV VP1 protein described herein comprise or consist of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type AAV VP 1 proteins or protein variants (e.g., such as an AAV1 VP1 protein, an AAV2 VP1 protein, an AAV3 VP1 protein, an AAV4 VP1 protein, an AAV5 VP1 protein, an AAV6 VP1 protein, an AAV7 VP1 protein, an AAV8 VP1 protein, an AAV9 VP1 protein, an AAV 10 VP1 protein, an AAV11 VP1 protein, an AAV 12 VP1 protein, or an AAV13 VP1 protein) described herein.

[0094] In some embodiments, the variants of the AAV VP2 protein described herein comprise or consist of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type AAV VP2 proteins or protein variants (e.g., such as an AAV1 VP2 protein, an AAV2 VP2 protein, an AAV3 VP2 protein, an AAV4 VP2 protein, an AAV5 VP2 protein, an AAV6 VP2 protein, an AAV7 VP2 protein, an AAV8 VP2 protein, an AAV9 VP2 protein, an AAV10 VP2 protein, an AAV11 VP2 protein, an AAV 12 VP2 protein, or an AAV13 VP2 protein) described herein.

[0095] In some embodiments, the variants of the AAV VP3 protein described herein comprise or consist of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%,WSGR Docket No. 60885-743.601at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type AAV VP3 proteins or protein variants (e.g., such as an AAV1 VP3 protein, an AAV2 VP3 protein, an AAV3 VP3 protein, an AAV4 VP3 protein, an AAV5 VP3 protein, an AAV6 VP3 protein, an AAV7 VP3 protein, an AAV8 VP3 protein, an AAV9 VP3 protein, an AAV10 VP3 protein, an AAV11 VP3 protein, an AAV 12 VP3 protein, or an AAV13 VP3 protein) described herein.

[0096] In some embodiments, the at least one viral protein described herein comprises at least one AAV helper protein. In some embodiments, the at least one AAV helper protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten AAV helper proteins. In some embodiments, the at least one AAV helper protein is an adenoviral helper protein.

[0097] In some embodiments, the adenoviral helper protein comprises a Human Adenovirus type 1 (HAdV-1) helper protein, a Human Adenovirus type 2 (HAdV-2) helper protein, a Human Adenovirus type 3 (HAdV-3) helper protein, a Human Adenovirus type 4 (HAdV-4) helper protein, a Human Adenovirus type 5 (HAdV-5) helper protein, a Human Adenovirus type 6 (HAdV-6) helper protein, a Human Adenovirus type 7 (HAdV-7) helper protein, a Human Adenovirus type 8 (HAdV-8) helper protein, a Human Adenovirus type 11 (HAdV-11) helper protein, a Human Adenovirus type 12 (HAdV-12) helper protein, a Human Adenovirus type 14 (HAdV-14) helper protein, a Human Adenovirus type 18 (HAdV-18) helper protein, a Human Adenovirus type 19 (HAdV-19) helper protein, a Human Adenovirus type 21 (HAdV-21) helper protein, a Human Adenovirus type 31 (HAdV-31) helper protein, a Human Adenovirus type 35 (HAdV-35) helper protein, a Human Adenovirus type 37 (HAdV-37) helper protein, a Human Adenovirus type 40 (HAdV-40) helper protein, a Human Adenovirus type 41 (HAdV-41) helper protein, a Human Adenovirus type 52 (HAdV-52) helper protein, a Human Adenovirus type 53 (HAdV-53) helper protein, and a Human Adenovirus type 64 (HAdV-64) helper protein, a variant thereof or any combination thereof.

[0098] In some embodiments, the variants of the adenoviral helper proteins described herein comprises or consists of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type adenoviral helper proteins or protein variants (e.g., such as HAdV-1, HAdV-2, HAdV-3, HAdV-4, HAdV-5, HAdV-6, HAdV-7, HAdV-8, HAdV-11, HAdV-12, HAdV-14, HAdV-18, HAdV-19, HAdV-21, HAdV-31, HAdV-35, HAdV-37, HAdV-40, HAdV-41, HAdV-52, HAdV-53, or HAdV-64) described herein.WSGR Docket No. 60885-743.601

[0099] In some embodiments, the adenoviral helper protein comprises an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2B protein, an adenovirus E4orf6 protein, a variant thereof, or any combination thereof.

[0100] In some embodiments, the variants of the adenoviral E1A protein described herein comprises or consists of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type adenoviral helper proteins or protein variants (e.g., such as HAdV-1 E1A protein, HAdV-2 E1A protein, HAdV-3 El A protein, HAdV-4 El A protein, HAdV-5 El A protein, HAdV-6 El A protein, HAdV-7 El A protein, HAdV-8 El A protein, HAdV-11 El A protein, HAdV-12 El A protein, HAdV-14 El A protein, HAdV-18 El A protein, HAdV-19 El A protein, HAdV-21 El A protein, HAdV-31 El A protein, HAdV-35 El A protein, HAdV-37 El A protein, HAdV-40 El A protein, HAdV-41 El A protein, HAdV-52 El A protein, HAdV-53 El A protein, orHAdV-64ElA protein) described herein.

[0101] In some embodiments, the variants of the adenoviral E1B protein described herein comprises or consists of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type adenoviral helper proteins or protein variants (e.g., such as HAdV-1 E1B protein, HAdV-2 E1B protein, HAdV-3 E1B protein, HAdV-4 E1B protein, HAdV-5 E1B protein, HAdV-6 E1B protein, HAdV-7 E1B protein, HAdV-8 E1B protein, HAdV-11 E1B protein, HAdV-12 E1B protein, HAdV-14 E1B protein, HAdV-18 E1B protein, HAdV-19 E1B protein, HAdV-21 E1B protein, HAdV-31 E1B protein, HAdV-35 E1B protein, HAdV-37 E1B protein, HAdV-40 E1B protein, HAdV-41 E1B protein, HAdV-52 E1B protein, HAdV-53 E1B protein, or HAdV-64 E1B protein) described herein.

[0102] In some embodiments, the variants of the adenoviral E2B protein described herein comprises or consists of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type adenoviral helper proteins or protein variants (e.g., such as HAdV-1 E2B protein, HAdV-2 E2B protein, HAdV-3 E2B protein, HAdV-4 E2B protein, HAdV-5 E2B protein, HAdV-6 E2B protein, HAdV-7 E2B protein, HAdV-8 E2B protein, HAdV-11 E2B protein, HAdV-12 E2B protein,WSGR Docket No. 60885-743.601HAdV-14 E2B protein, HAdV-18 E2B protein, HAdV-19 E2B protein, HAdV-21 E2B protein, HAdV-31 E2B protein, HAdV-35 E2B protein, HAdV-37 E2B protein, HAdV-40 E2B protein, HAdV-41 E2B protein, HAdV-52 E2B protein, HAdV-53 E2B protein, or HAdV-64 E2B protein) described herein.

[0103] In some embodiments, the variants of the adenoviral E4orf6 protein described herein comprises or consists of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type adenoviral helper proteins or protein variants (e.g., such as HAdV-1 E4orf6 protein, HAdV-2 E4orf6 protein, HAdV-3 E4orf6 protein, HAdV-4 E4orf6 protein, HAdV-5 E4orf6 protein, HAdV-6 E4orf6 protein, HAdV-7 E4orf6 protein, HAdV-8 E4orf6 protein, HAdV-11 E4orf6 protein, HAdV-12 E4orf6 protein, HAdV-14 E4orf6 protein, HAdV-18 E4orf6 protein, HAdV-19 E4orf6 protein, HAdV-21 E4orf6 protein, HAdV-31 E4orf6 protein, HAdV-35 E4orf6 protein, HAdV-37 E4orf6 protein, HAdV-40 E4orf6 protein, HAdV-41 E4orf6 protein, HAdV-52 E4orf6 protein, HAdV-53 E4orf6 protein, or HAdV-64 E4orf6 protein) described herein.

[0104] In some embodiments, the at least one exogenous nucleic acid described herein comprises a nucleic acid sequence encoding an adenoviral virus-associated (VA) RNA, or a variant thereof.

[0105] In some embodiments, the variants of the adenoviral VA RNA described herein comprises or consists of an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the wild-type adenoviral helper proteins or protein variants (e.g., such as HAdV-1 VARNA, HAdV-2 VARNA, HAdV-3 VA RNA, HAdV-4 VA RNA, HAdV-5 VA RNA, HAdV-6 VA RNA, HAdV-7 VA RNA, HAdV-8 VA RNA, HAdV-11 VA RNA, HAdV-12 VA RNA, HAdV-14 VA RNA, HAdV-18 VA RNA, HAd V- 19 VA RNA, HAdV-21 VA RNA, HAdV-31 VA RNA, HAdV-35 V A RNA, HAdV-37 VA RNA, HAdV-40 VA RNA, HAdV-41 VA RNA, HAdV-52 VA RNA, HAdV-53 VA RNA, or HAdV-64 VA RNA) described herein.

[0106] In some embodiments, the at least one promoter described herein comprises an inducible promoter. In some embodiments, the at least one promoter described herein comprises a constitutive promoter.

[0107] In some embodiments, the at least one exogenous nucleic acid described herein comprises a second nucleic acid sequence. In some embodiments, the at least one exogenousWSGR Docket No. 60885-743.601nucleic acid described herein comprises a second nucleic acid sequence, wherein the second nucleic encodes a light-controllable transcriptional regulator described herein.

[0108] In some embodiments, the method described herein uses optogenetics for (e.g., temporally and / or spatially) controlling the expression of one or more viral proteins. For instance, in some embodiments, a promoter regulating (e.g., expression of) one or more viral proteins may be controlled by light. In some embodiments, the method described herein involves the use of a light-controllable transcriptional regulator which can either bind to a promoter (or a region adjacent to or near a promoter), or dissociate from a promoter (or a region adjacent to or near a promoter), to activate expression of the one or more viral proteins. In some embodiments, the light-controllable transcriptional regulator is a transcriptional regulator fused to a light-controllable domain (e.g., an optical switch). In some embodiments, the light-controllable transcriptional regulator is a light-controllable transcriptional activator. The light-controllable transcriptional activator can be a transcriptional activator fused to a light-controllable domain (e.g., an optical switch).

[0109] In some embodiments, the light-controllable transcriptional regulator is a light-controllable transcriptional repressor. The light-controllable transcriptional repressor can be a transcriptional repressor fused to a light-controllable domain. Advantageously, the method described herein does not lead to permanent expression of the one or more viral proteins, and expression of the one or more viral proteins may be turned on or off simply by supplying or removing light, as described herein.

[0110] In some embodiments, the method described herein involves the use of one or more recombinases. In some embodiments, the method described herein involves the use of one or more light-controllable recombinases. A recombinase recognizes a specific DNA sequence, and if there are two recognition sequences in the proper arrangement, it can excise or flip the orientation of the DNA between the two sites. By briefly activating the recombinase, a permanent change can be made to the DNA, which offers the prospect of permanently switching expression of one or more viral proteins with only a short activation phase.

[0111] In some embodiments, the at least one exogenous nucleic acid described herein comprises a second nucleic acid sequence encoding a light-controllable transcriptional regulator. In some embodiments, the method described herein further comprises expressing the light-controllable transcriptional regulator.

[0112] In some cases, the light-controllable transcriptional regulator is a light-controllable transcriptional activator. The light-controllable transcriptional activator may be a transcriptional activator that is fused to or otherwise associated with a light-controllable domain, such as an optical switch or a photostate-dependent interaction partner of the optical switch (e.g., as describedWSGR Docket No. 60885-743.601herein). In some cases, the at least one light-controllable transcriptional regulator may be a light-controllable transcriptional repressor. The light-controllable transcriptional repressor may be a transcriptional repressor that is fused to or otherwise associated with a light-controllable domain, such as an optical switch or a photostate-dependent interaction partner of the optical switch (e.g., as described herein).

[0113] In various aspects, the light-controllable transcriptional regulator is a light-controllable transcriptional activator. In one embodiment, the light-controllable transcriptional activator, upon illumination with or removing of light (e.g., at a particular wavelength or wavelength range), binds to or otherwise associates with a promoter sequence (e.g., an inducible promoter), thereby inducing expression of one or more viral proteins.

[0114] FIGS. 1A, IB, 2A, 2B, 3A and 3B depict various exemplary embodiments that use a light-controllable activator or suppressor to control expression of one or more viral proteins described herein. As shown in FIG. 1A, in one embodiment, a transcriptional activator is fused to a first light-heterodimerizing domain (e.g., light-heterodimerizing domain 1). A second light-heterodimerizing domain (e.g., light-heterodimerizing domain 2), which is capable of dimerizing with the first light-heterodimerizing domain, is fused to a DNA-binding domain. The DNA-binding domain binds to a DNA sequence within or near the promoter sequence (e.g. , or binds to a region functionally associated with the promoter sequence). In the absence of light, the first and second light-heterodimerizing domains are not associated and expression of the one or more viral proteins is turned off. Upon illumination with light (e.g., at an appropriate wavelength or wavelength range), the first and second light-heterodimerizing domains heterodimerize, bringing the transcriptional activator into close contact with the inducible promoter. The transcriptional activator then induces activity of the inducible promoter, and the one or more viral proteins are expressed. In this scenario, those cells that are illuminated with light express and produce the one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, do not express and produce the one or more viral proteins. In some cases, removal of light or illumination with light at a different wavelength or wavelength range can reverse the expression (e.g., the expression is turned off). In some cases, removal of light (e.g., removal of light at a first wavelength or wavelength range) or absence of light can initiate expression and production of the one or more viral proteins. For example, upon removing light (e.g., absences of light or removing light at an appropriate wavelength or wavelength range), the first and second light-heterodimerizing domains can heterodimerize, bringing the transcriptional activator into close contact with the inducible promoter. The transcriptional activator then induces activity of the inducible promoter, and the protein the one or more viral proteins are expressed.WSGR Docket No. 60885-743.601

[0115] In another embodiment, as depicted in FIG. IB, a transcriptional activator is fused to a light-homodimerizing domain and a DNA-binding domain. In this scenario, the DNA-binding domain, upon homodimerizing with another DNA-binding domain, binds to a DNA sequence within or near the promoter sequence (e.g., or binds to a region functionally associated with the promoter sequence). Upon illumination with light (e.g., at an appropriate wavelength or wavelength range), the light-homodimerizing domains homodimerize, which brings two DNA-binding domains into close contact such that they homodimerize. Upon homodimerizing, the DNA-binding domain binds to the promoter sequence bringing the transcriptional activator into close contact with the inducible promoter (e.g., or binds to a region functionally associated with the promoter sequence). The transcriptional activator then induces activity of the inducible promoter, and the one or more viral proteins are expressed. In this scenario, those cells that are illuminated with light express and produce the one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, do not express and produce the one or more viral proteins. In some cases, removal of light or illumination with light at a different wavelength or wavelength range promotes reversion (e.g., the two light-homodimerizing domains dissociate and expression of the one or more viral proteins is turned off). In various aspects, the light-controllable transcriptional activator, in the absence of light or in the absence of light at a particular wavelength, binds to or associates with a promoter sequence (e.g., or binds to a region functionally associated with the promoter sequence), thereby inducing expression of the one or more viral proteins. In the presence of light, the light-controllable transcriptional activator does not bind to or it dissociates from the promoter sequence (e.g., an inducible promoter), thereby preventing or reducing expression of the one or more viral proteins.

[0116] As shown in FIG. 2A, in one embodiment, a transcriptional activator is fused to a first light-dissociating domain (e.g., light-dissociating domain 1). A second light-dissociating domain (e.g., light-dissociating domain 2) is fused to a DNA-binding domain. The DNA-binding domain binds to a DNA sequence within or near the promoter sequence (e.g., or binds to a region functionally associated with the promoter sequence). In the absence of light, the first and second light-dissociating domains are associated, bringing the transcriptional activator in close contact with the inducible promoter, and expression of the one or more viral proteins is turned on. Upon illumination with light (e.g., at an appropriate wavelength or wavelength range), the first and second light-dissociating domains dissociate, releasing the transcriptional activator from the inducible promoter thereby turning off expression. In this scenario, those cells that are illuminated with light do not express and produce the one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, express and and produce the one or more viral proteins. In some cases, removal of light or illumination withWSGR Docket No. 60885-743.601light at a different wavelength or wavelength range promotes reversion (e.g., the two lightdissociating domains associate and expression of the one or more viral proteins is turned on).

[0117] In another embodiment, as depicted in FIG. 2B, a transcriptional activator is fused to a light-dissociating domain and a DNA-binding domain. In this scenario, the DNA-binding domain, upon homodimerizing with another DNA-binding domain, binds to a DNA sequence within or near the promoter sequence (e.g., or binds to a region functionally associated with the promoter sequence). In the absence of light, the light-dissociating domain is homodimerized with another light-dissociating domain, allowing the DNA-binding domain to homodimerize and bind the inducible promoter, and bringing the transcriptional activator into close contact with the inducible promoter. The transcriptional activator then induces activity of the inducible promoter, and the one or more viral proteins are expressed. Upon illumination with light (e.g., at an appropriate wavelength or wavelength range), the light-dissociating domains dissociate, which releases the transcriptional activator from the inducible promoter and turns off expression of the one or more viral proteins. In this scenario, those cells that are illuminated with light do not express the one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, express the one or more viral proteins. In some cases, removal of light or illumination with light at a different wavelength or wavelength range promotes reversion (e.g., the two light-dissociating domains associate and expression of the one or more viral proteins is turned on).

[0118] In another embodiment, as depicted in FIG. 3A, a fusion protein is provided which comprises a transcriptional control module and a light-sensitive subcellular localization module. The transcriptional control module, such as depicted in FIG. 3A, may comprise a DNA binding domain fused to a transcriptional activator. The light-sensitive subcellular localization module, such as depicted in FIG.3A, may comprise a light-responsive domain (e.g., a protein that changes shape or undergoes a conformational change in response to light; “light uncaging domain” in FIG.3A) and a nuclear import signal. As shown in FIG. 3A, the light-responsive domain may, in the absence of light, be in a closed configuration such that the nuclear import signal is caged. Also depicted in FIG.3A, the fusion protein may further comprise a nuclear export signal, which is not caged, such that when the nuclear import signal is caged, the fusion protein is exported from the nucleus into the cytoplasm. In the absence of light, the light-responsive domain (e.g., “light uncaging domain” in FIG. 3A) is in a closed conformation such that the nuclear import signal is inaccessible, and the nuclear export signal is exposed. In this scenario, the DNA-binding domain and transcriptional activator is localized in the cytoplasm of the cell. In the presence of light, the light-responsive domain (e.g., “light uncaging domain” in FIG. 3A) undergoes a conformational change such that the nuclear import signal is exposed allowing the transcriptional activator to beWSGR Docket No. 60885-743.601imported from the cytoplasm into the nucleus. The DNA-binding domain then binds to a DNA sequence recognized by the DNA-binding domain that is within or near a promoter sequence and brings the transcriptional activator into close contact with the promoter (e.g., or binds to a region functionally associated with the promoter sequence). The transcriptional activator then induces activity of the (e.g, inducible) promoter, and the one or more viral proteins are expressed. In this scenario, those cells that are illuminated with light express the one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, do not express the one or more viral proteins. In some cases, expression of the one or more viral proteins can be reverted by, e.g, illuminating the cell with light at a different wavelength of light or by turning off illumination completely (e.g, exposing the cell to dark) such that the light-responsive domain undergoes a conformational change to a closed position thereby caging the nuclear import signal and promoting export of the fusion protein from the nucleus to the cytoplasm (and repression or turning off expression of the one or more viral proteins).

[0119] In another embodiment, as depicted in FIG. 3B, a fusion protein is provided which comprises a transcriptional control module and a light-sensitive subcellular localization module. The transcriptional control module, such as depicted in FIG. 3B, may comprise a DNA binding domain fused to a transcriptional activator. The light-sensitive subcellular localization module, such as depicted in FIG.3B, may comprise a light-responsive domain (e.g., a protein that changes shape or undergoes a conformational change in response to light; “light uncaging domain” in FIG.3B) and a nuclear export signal. As shown in FIG. 3B, the light-responsive domain may, in the absence of light, be in a closed configuration such that the nuclear export signal is caged. Also depicted in FIG. 3B, the fusion protein may further comprise a nuclear import signal, which is not caged, such that when the nuclear export signal is caged, the fusion protein is imported from the cytoplasm into the nucleus. In the absence of light, the light-responsive domain (e.g., “light uncaging domain” in FIG. 3B) is in a closed conformation such that the nuclear export signal is inaccessible, and the nuclear import signal is exposed. In this scenario, the DNA-binding domain and transcriptional activator is localized in the nucleus of the cell. The DNA-binding domain then binds to a DNA sequence recognized by the DNA-binding domain that is within or near a promoter sequence and brings the transcriptional activator into close contact with the promoter (e.g., or binds to a region functionally associated with the promoter sequence). The transcriptional activator then induces activity of the inducible promoter, and the one or more viral proteins are expressed. In the presence of light, the light-responsive domain (e.g., “light uncaging domain” in FIG. 3B) undergoes a conformational change such that the nuclear export signal is exposed allowing the transcriptional activator to be exported from the nucleus into the cytoplasm. In this scenario, those cells that are illuminated with light do not express the one or more viral proteins,WSGR Docket No. 60885-743.601whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, express the one or more viral proteins. In some cases, non-expression of the one or more viral proteins can be reverted by, e.g., illuminating the cell with light at a different wavelength of light or by turning off illumination completely (e.g., exposing the cell to dark) such that the light-responsive domain undergoes a conformational change to a closed position thereby caging the nuclear export signal and promoting import of the fusion protein from the cytoplasm to the nucleus (and induction of expression and production of the one or more viral proteins).

[0120] In various aspects, the light-controllable transcriptional regulator is a light-controllable transcriptional repressor. In one embodiment, the light-controllable transcriptional repressor, upon illumination with or removing of light (e.g., at a particular wavelength or wavelength range), binds to or otherwise associates with a promoter sequence (e.g., a constitutive promoter, or a sequence between a constitutive promoter and a transcriptional start site; binds to a region functionally associated with the promoter sequence), thereby repressing the constitutive promoter and preventing or reducing expression of the one or more viral proteins.

[0121] FIGS. 4A, 4B, 5A, 5B, 6A, and 6B depict various embodiments that use a light-controllable transcriptional repressor to control expression of the one or more viral proteins. As shown in FIG. 4A, in one embodiment, a transcriptional repressor is fused to a first light-heterodimerizing domain (e.g., light-heterodimerizing domain 1). A second light-heterodimerizing domain (e.g., light-heterodimerizing domain 2), which is capable of dimerizing with the first light-heterodimerizing domain, is fused to a DNA-binding domain. The DNA-binding domain binds to a DNA sequence within or near a constitutive promoter sequence (e.g. , a region functionally associated with the promoter sequence). In the absence of light, the first and second light-heterodimerizing domains are not associated and expression of the one or more viral proteins is turned on (controlled by the constitutive promoter). Upon illumination with light (e.g., at an appropriate wavelength or wavelength range), the first and second light-heterodimerizing domains dimerize, bringing the transcriptional repressor into close contact with the constitutive promoter sequence or a sequence between the constitutive promoter and the transcriptional start site. The transcriptional repressor blocks activity of the constitutive promoter and expression of the one or more viral proteins is turned off. In this scenario, those cells that are illuminated with light do not express the one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, express the one or more viral proteins. In some cases, removal of light or illumination with light at a different wavelength than what was used to repress expression of the one or more viral proteins, promotes reversion (e.g., the two light-heterodimerizing domains dissociate and expression of the one or more viral proteins is turned on).WSGR Docket No. 60885-743.601

[0122] In another embodiment, as depicted in FIG. 4B, a transcriptional repressor is fused to a light-homodimerizing domain and a DNA-binding domain. In this scenario, the DNA-binding domain, upon homodimerizing with another DNA-binding domain, binds to a DNA sequence within or near a constitutive promoter sequence (e.g., a region functionally associated with the promoter sequence). In the absence of light, the first and second light-homodimerizing domains are not associated and expression of the one or more viral proteins is turned on (controlled by the constitutive promoter). Upon illumination with light (e.g., at an appropriate wavelength or wavelength range), the first and second light-homodimerizing domains dimerize, bringing the transcriptional repressor into close contact with the constitutive promoter sequence or a sequence between the constitutive promoter and the transcriptional start site. The transcriptional repressor blocks activity of the constitutive promoter and expression of the one or more viral proteins is turned off. In this scenario, those cells that are illuminated with light do not express the one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, express the one or more viral proteins. In some cases, removal of light or illumination with light at a different wavelength than what was used to repress expression of the one or more viral proteins can be used to promote reversion (e.g., the two light-homodimerizing domains dissociate and expression of the one or more viral proteins is turned on).

[0123] In various aspects, the light-controllable transcriptional repressor, in the absence of light, binds to or associates with a constitutive promoter sequence (e.g., a constitutive promoter, or a sequence between a constitutive promoter and a transcriptional start site; a region functionally associated with the promoter sequence), thereby preventing or reducing expression of the one or more viral proteins as described herein. In the presence of light, the light-controllable transcriptional repressor does not bind to or dissociates from the constitutive promoter sequence (e.g., a constitutive promoter, or a sequence between a constitutive promoter and a transcriptional start site), thereby activating expression of the one or more viral proteins.

[0124] As shown in FIG. 5A, in one embodiment, a transcriptional repressor is fused to a first light-dissociating domain (e.g., light-dissociating domain 1). A second light-dissociating domain (e.g., light-dissociating domain 2) is fused to a DNA-binding domain. The DNA-binding domain binds to a DNA sequence within or near a constitutive promoter sequence (e.g., a region functionally associated with the promoter sequence). In the absence of light, the first and second light-dissociating domains are associated, bringing the transcriptional repressor in close contact with the constitutive promoter sequence or a sequence between the constitutive promoter and the transcriptional start site, and expression of the one or more viral proteins is turned off. Upon illumination with light (e.g., at an appropriate wavelength or wavelength range), the first and second light-dissociating domains dissociate, releasing the transcriptional repressor from theWSGR Docket No. 60885-743.601constitutive promoter sequence thereby turning on expression of the one or more viral proteins. In this scenario, those cells that are illuminated with light express one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, do not express one or more viral proteins. In some cases, removal of light or illumination with light at a different wavelength or wavelength range promotes reversion (e.g. , the two light-dissociating domains associate and expression of the one or more viral proteins is turned off).

[0125] In another embodiment, as depicted in FIG. 5B, a transcriptional repressor is fused to a light-dissociating domain and a DNA-binding domain. In this scenario, the DNA-binding domain, upon homodimerizing with another DNA-binding domain, binds to a DNA sequence within or near a constitutive promoter sequence (e.g., a region functionally associated with the promoter sequence). In the absence of light, the light-dissociating domain is homodimerized with another light-dissociating domain, allowing the DNA-binding domain to homodimerize and bind the constitutive promoter sequence or a sequence between the constitutive promoter and the transcriptional start site, and bringing the transcriptional repressor into close contact with the constitutive promoter sequence or a sequence between the constitutive promoter and the transcriptional start site. The transcriptional repressor blocks activity of the constitutive promoter and expression of the one or more viral proteins is turned off. Upon illumination with light (e.g., at an appropriate wavelength or wavelength range), the light-dissociating domains dissociate, which releases the transcriptional repressor from the constitutive promoter or a sequence between the constitutive promoter and the transcriptional start site, and expression of the one or more viral proteins is turned on. In this scenario, those cells that are illuminated with light express one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, do not express one or more viral proteins. In some cases, removal of light or illumination with light at a different wavelength than what was used to induce expression of one or more viral proteins, promotes reversion (e.g., the two light-dissociating domains associate and expression of the one or more viral proteins is turned off).

[0126] In another embodiment, as depicted in FIG. 6A, a fusion protein is provided which comprises a transcriptional control module and a light-sensitive subcellular localization module. The transcriptional control module, such as depicted in FIG. 6A, may comprise a DNA binding domain fused to a transcriptional repressor. The light-sensitive subcellular localization module, such as depicted in FIG. 6A, may comprise a light-responsive domain (e.g., a protein that changes shape or undergoes a conformational change in response to light; “light uncaging domain” in FIG.6A) and a nuclear import signal. As shown in FIG. 6A, the light-responsive domain may, in the absence of light, be in a closed configuration such that the nuclear import signal is caged. AlsoWSGR Docket No. 60885-743.601depicted in FIG. 6A, the fusion protein may further comprise a nuclear export signal, which is not caged, such that when the nuclear import signal is caged, the fusion protein is exported from the nucleus into the cytoplasm. In the absence of light, the light-responsive domain (e.g., “light uncaging domain” in FIG. 6A) is in a closed conformation such that the nuclear import signal is inaccessible, and the nuclear export signal is exposed. In this scenario, the DNA-binding domain and transcriptional repressor is localized in the cytoplasm of the cell. In the presence of light, the light-responsive domain (e.g., “light uncaging domain” in FIG. 6A) undergoes a conformational change such that the nuclear import signal is exposed allowing the transcriptional repressor to be imported from the cytoplasm into the nucleus. The DNA-binding domain then binds to a DNA sequence recognized by the DNA-binding domain that is within or near a constitutive promoter sequence and brings the transcriptional repressor into close contact with the constitutive promoter or a sequence between the constitutive promoter and the transcriptional start site (e.g., a region functionally associated with the promoter sequence). The transcriptional repressor then represses activity of the constitutive promoter, and the one or more viral proteins are not expressed. In this scenario, those cells that are illuminated with light do not express the one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, express one or more viral proteins. In some cases, repression of expression of the one or more viral proteins can be reverted by, e.g., illuminating the cell with light at a different wavelength of light or by turning off illumination completely (e.g., exposing the cell to dark) such that the light-responsive domain undergoes a conformational change to a closed position thereby caging the nuclear import signal and promoting export of the fusion protein from the nucleus to the cytoplasm (and induction of expression of the one or more viral proteins).

[0127] In another embodiment, as depicted in FIG. 6B, a fusion protein is provided which comprises a transcriptional control module and a light-sensitive subcellular localization module. The transcriptional control module, such as depicted in FIG. 6B, may comprise a DNA binding domain fused to a transcriptional repressor. The light-sensitive subcellular localization module, such as depicted in FIG.6B, may comprise a light-responsive domain (e.g., a protein that changes shape or undergoes a conformational change in response to light; “light uncaging domain” in FIG.6B) and a nuclear export signal. As shown in FIG. 6B, the light-responsive domain may, in the absence of light, be in a closed configuration such that the nuclear export signal is caged. Also depicted in FIG. 6B, the fusion protein may further comprise a nuclear import signal, which is not caged, such that when the nuclear export signal is caged, the fusion protein is imported from the cytoplasm into the nucleus. In the absence of light, the light-responsive domain (e.g., “light uncaging domain” in FIG. 6B) is in a closed conformation such that the nuclear export signal isWSGR Docket No. 60885-743.601inaccessible, and the nuclear import signal is exposed. In this scenario, the DNA-binding domain and transcriptional repressor is localized in the nucleus of the cell. The DNA-binding domain then binds to a DNA sequence recognized by the DNA-binding domain that is within or near a constitutive promoter sequence and brings the transcriptional repressor into close contact with the constitutive promoter or a sequence between the constitutive promoter and the transcriptional start site (e.g., a region functionally associated with the promoter sequence). The transcriptional repressor then represses activity of the constitutive promoter, and the one or more viral proteins are not expressed. In the presence of light, the light-responsive domain (e.g., “light uncaging domain” in FIG. 6B) undergoes a conformational change such that the nuclear export signal is exposed allowing the transcriptional repressor to be exported from the nucleus into the cytoplasm. In this scenario, those cells that are illuminated with light express the one or more viral proteins, whereas those cells that are not illuminated with light, or not illuminated with light at the appropriate wavelength, do not express the one or more viral proteins. In some cases, expression of the one or more viral proteins can be reverted by, e.g., illuminating the cell with light at a different wavelength of light or by turning off illumination completely (e.g., exposing the cell to dark) such that the light-responsive domain undergoes a conformational change to a closed position thereby caging the nuclear export signal and promoting import of the fusion protein from the cytoplasm to the nucleus (and repression or turning off expression of the one or more viral proteins).

[0128] In some embodiments, the methods described herein use light-controllable recombinases (e.g., light-activatable recombinases or light-induced expression of constitutively active recombinases) that are controlled by light in order to control expression of the at least one gene encoding the one or more viral proteins. In some embodiments, the methods described herein comprises introducing or providing at least one mammalian cell comprising (1) a nucleic acid sequence comprising at least one gene encoding the one or more viral proteins and (2) an expression control element that controls expression of the at least one gene. In some embodiments, the expression control element comprises light-controllable recombinases.

[0129] In other aspects of the disclosure, control of the expression of the at least one gene encoding the one or more viral proteins may involve the use of a light-controllable recombinase. In such scenarios, expression of the at least one gene may be under the control of a (e.g., constitutive) promoter. A blocking sequence may be inserted between the (e.g., constitutive) promoter and the sequence encoding the at least one gene encoding the one or more viral proteins, such that expression of the gene is blocked, when the blocking sequence is present. Upon removal of the blocking sequence, expression of the at least one gene encoding the one or more viral proteins is activated. Removal of the blocking sequence may be achieved by use of a recombinaseWSGR Docket No. 60885-743.601(e.g., a light-controllable recombinase). In such scenarios, the blocking sequence is flanked by recombinase recognition sites, such that upon illumination with light, the light-controllable (or light-activatable) recombinase excises the blocking sequence, thereby allowing expression of the at least one gene encoding the one or more viral proteins. By fusing the recombinase to a light-controllable domain (e.g., an optogenetic switch as described herein), the recombinase can be activated by light (e.g., at a particular wavelength or wavelength range). In such scenarios, illumination of the at least one mammalian cell leads to the expression of the at least one gene encoding the one or more viral proteins. In another embodiment, the recombinase fused to a light-controllable domain (e.g., an optogenetic switch as described herein) can be deactivated by light (e.g., at a particular wavelength or wavelength range). In such scenarios, removal of light (or removal of light at a specific wavelength or wavelength range) or illumination with light at a different wavelength or wavelength range of the at least one mammalian cell leads to the expression of the at least one gene encoding the one or more viral proteins.

[0130] FIG. 7 depicts a non-limiting example of a nucleic acid cassette suitable for use with the disclosure herein. A promoter 703 may be operably linked to a nucleic acid sequence comprising at least one gene encoding the one or more viral proteins. In some cases, a blocking sequence 702 may be located between the promoter sequence and the nucleic acid sequence comprising at least one gene encoding the one or more viral proteins (e.g., wherein the blocking sequence is downstream of the promoter). A blocking sequence may be any nucleic acid sequence that prevents transcription of the nucleic acid sequence encoding the one or more viral proteins, such as a nucleic acid sequence encoding one or more stop codons and / or transcription terminator sequences. In some cases, the blocking sequence may comprise an expression cassette or multiple expression cassettes, each containing a transcription terminator sequence. In some cases, the blocking sequence may be flanked by recombinase recognition sites 701 (e.g., wherein the recombinase recognition sites are recognized by a light-controllable recombinase). In such scenarios, in the presence of an activated recombinase, the blocking sequence is excised allowing for expression of the at least one gene encoding the one or more viral proteins. In some cases, the recombinase may be activatable, e.g., by light, as described herein, thus allowing for control of recombinase activity. In such cases, the recombinase may be expressed in the cell but may be in an inactive state until the at least one mammalian cell is exposed to light at a particular wavelength or wavelength range. Upon exposure of the at least one mammalian cell to light at a particular wavelength or wavelength range, the recombinase may be activated, leading to excision of the blocking sequence and expression of the one or more viral proteins. In another embodiment, a constitutively active recombinase is expressed in a light-dependent manner (e.g. with methods as described in, for example, FIGS. 1-6). In some cases, expression of the recombinase is inducedWSGR Docket No. 60885-743.601by light. In some cases, expression of the recombinase is induced by removal of light (or removal of light at a specific wavelength or wavelength range) or illumination with light at a different wavelength or wavelength range.

[0131] In an alternative embodiment, rather than excision of a blocking sequence, the light-controllable recombinase may be used to flip nucleic acid sequences such that the nucleic acid sequences are under the control of a promoter, thereby resulting in expression of the one or more viral proteins.

[0132] In some cases, more than one recombinase may be used, such that, depending upon which recombinase is activated, a different viral protein is expressed. For example, a cell may express more than one light-controllable recombinase (e.g., each fused to a different light-controllable domain or expressed under a different light-inducible promoter). When expression of viral protein A is desired, the cell may be exposed to light at a first wavelength or wavelength range, thereby activating or expressing a first recombinase, thereby resulting in expression of first viral protein(s). When expression of viral protein B is desired, the cell may be exposed to light at a second, different wavelength or wavelength range, thereby activating or expressing a second recombinase, thereby resulting in expression of second viral protein(s). In a population of cells, this method may be employed to (e.g., temporally) control expression of different viral protein(s).

[0133] In some instances, the recombinase may be activated using a light-activatable system. The light-activatable system may be as described in Table 1.Table 1. Light-Activatable systems

[0134] In various aspects, a combination of light-controllable domains (e.g., a first light-controllable domain and a second light-controllable domain) may be used (e.g., each of the light-controllable domains may be fused to a portion of the recombinase). In some cases, the first light-WSGR Docket No. 60885-743.601controllable domain and the second light-controllable domain can be binding partners, such that upon illumination with light at a particular wavelength or within a particular wavelength range, the first and second light-controllable domains heterodimerize or hetero-oligomerize. The first and second light-controllable domains, upon illumination with light at a particular wavelength or within a particular wavelength range, heterodimerize or hetero-oligomerize, thereby bringing the protein domains (or functional domains or functional portions thereof) into close contact with one another such that the recombinase(s) is / are activated. In some cases, removal of light or illumination with light at a different wavelength or wavelength range induces the interaction between the first light-controllable domain and the second light-controllable domain. In some cases, the first light-controllable domain and the second light-controllable domain are identical.

[0135] In various aspects, the light-controllable domain comprises a Light-Oxygen- Voltage (LOV) photoreceptor domain, a LOV2 photoreceptor domain, a Cryptochrome (CRY) domain, Blue-light-using FAD (BLUF) photoreceptor domain, a Phytochrome (PHY) domain, CIB1 (cryptochrome-interacting basic-helix-loop-helix protein 1) (or a functional portion or domain thereof; e.g., CIBN (N-terminal domain of CIB 1)), a PIF (phytochrome interacting factor) domain, a Dronpa domain, a UVR8 photoreceptor domain, a COP1 domain, a BphPl domain, a QPAS-1 domain, a cobalamin-binding domain (CBD), or a combination thereof. For example, as described in FIGS. 1-6, the light controllable domain described herein can be operably coupled to a transcription regulator e.g., a transcriptional activator or transcriptional repressor described herein).

[0136] In some instances, a combination of light-controllable domains is used, wherein the first light-controllable domain is cryptochrome 2 (or a variant or a functional portion thereof) and the second light-controllable domain is CIB1 (or a variant or a functional portion thereof; e.g., CIBN). In some instances, a combination of light-controllable domains is used, wherein the first light-controllable domain is BphPl (or a variant or a functional portion thereof) and the second light-controllable domain is QPAS1 (or a variant or a functional portion thereof). In some cases, the light-controllable domain (or combination of light-activatable domains) is selected from Table 2. In some cases, the light-controllable domain may have an amino acid sequence having at least about 50% sequence identity (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater) to any one of the light-controllable domains described in Table 2. The light-controllable domains can be either a photoreceptor or a photostate-dependent interaction partner of the photoreceptor.WSGR Docket No. 60885-743.601Table 2. Non-limiting examples of light-controllable domain systemsWSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601

[0137] In some cases, the methods described herein may act via an intermediate transcriptional regulatory protein or gene circuit, for example an amplifier circuit. In such cases, the methods described herein may modulate the expression of for example a constitutively active transcriptional activator or repressor that interacts with a promoter operatively coupled to a nucleotide sequence that encodes one or more viral proteins. In such cases the methods and composition provided herein may ultimately modulate the expression of the at least one gene encoding the protein of interest (e.g., viral proteins) without directly interacting with the promoter of the at least one gene.

[0138] In another aspect of the method described herein, the recombinase may be activated by a specific wavelength or wavelength range of light. The recombinase may be constitutively expressed in an inactive form. The recombinase may be conditionally expressed by a light-induced system in an inactive form. The recombinase may mediate irreversible excision. The recombinase may be a serine integrase, such as $C31, TP901, and Bxbl. The recombinase may be a tyrosine recombinase, such as Cre, VCre and Flp.

[0139] In another embodiment, dimerization and activation of the recombinase can be induced by light (e.g., optogenetic dimerization). The first half of the recombinase and the second half of the recombinase may be fused to a light-controllable domain. In various aspects, the light-controllable domain can comprise a Light-Oxygen- Voltage (LOV) photoreceptor domain, a LOV2 photoreceptor domain, a Cryptochrome (CRY) domain, Blue-light-using FAD (BLUF) photoreceptor domain, a Phytochrome (PHY) domain, CIB1 (cryptochrome-interacting basic-helix-loop-helix protein 1) (or a functional portion or domain thereof; e.g., CIBN), a PIF (phytochrome interacting factor) domain, a Dronpa domain, a UVR8 photoreceptor domain, a COP1 domain, a BphPl domain, a QPAS-1 domain, a cobalamin-binding domain (CBD), or a combination thereof. Regulation of the dimerization and activation of the recombinase may utilize any of the light-controllable systems described in Table 2. Dimerization and activation may alternatively be induced by temperature.

[0140] In various aspects, the method described herein can involve exposing the cells (e.g., genetically engineered to express the fusion protein comprising the recombinase and the light-controllable domain) with light at a particular wavelength or light within a particular wavelength range. The wavelength of light can be selected such that the light is capable of activating the light-controllable domain. For example, Table 2 provides non-limiting examples of light parameters for different light-controllable domain systems. The wavelength of light may be one or more of infrared, near infrared, visible light (e.g., red, green, blue), ultraviolet light, or a combination thereof. Infrared light may comprise light at a wavelength of about 780 nm to 1 mm. Near infrared light may comprise light at a wavelength of about 740 nm to about 780 nm. Red light mayWSGR Docket No. 60885-743.601comprise light at a wavelength of about 620 nm to 750 nm, 600 nm to 690 nm, or about 650 nm. Green light may comprise light at a wavelength of about 577 nm to about 492 nm. Blue light may comprise light at a wavelength of 492 to about 455 nm, or about 440 nm to about 473 nm. Ultraviolet light may comprise light from about 10 nm to 400 nm, or from about 280 to 315 nm. In various aspects, the wavelength of light is from 100 nm to 1 mm.

[0141] In some embodiments, the method described herein involves illuminating cells with light at a particular intensity or range of intensities. For example, the methods provided herein may involve illuminating cells with light at an intensity of about 2 pW / mm2In other examples, the methods provided herein may involve illuminating cells with light at an intensity of about 200 pW / mm2In some cases, the methods provided herein may involve illuminating cells with light at an intensity from about 2 pW / mm2to about 200 pW / mm2. It should be understood that the level of light intensity may vary and may depend on the type of optogenetic switch used and the duty cycle of illumination.

[0142] In some embodiments, the method described herein involves illuminating cells with light in an illumination pattern, such as a pulsing pattern. For example, the methods may involve illuminating cells for a period of time, turning off illumination of the cells for a period of time, and then repeating the on / off cycle for a number of times.

[0143] In another embodiment, the recombinase comprises a single chain polypeptide. The single chain polypeptide may be fused to a light-controllable domain to create a light-controllable recombinase. Exposure to light at a particular wavelength may result in activity of the recombinase. For instance, illumination of the AsLOV2-based Cre system LiCre with blue light results in activation of the recombinase.

[0144] In another embodiment, the recombinase may be fused to a PhoCl protein or a derivative thereof. Illumination with violet light (about 400 nm) results in cleavage of the PhoCl. In some instances, a PhoCl domain may be present in the fusion protein between a blocker domain and the recombinase domain. The blocker domain may be any domain that prevents the recombinase from functioning, such as a domain that prevents the recombinase from entering the nucleus. For instance, the blocker domain may be a steroid receptor domain which interacts with Hsp90 to prevent nuclear entry of the recombinase. Exposing the fusion protein comprising the blocker domain, the PhoCl domain, and the recombinase to violet light may result in cleavage of the PhoCl domain, allowing the recombinase to enter the nucleus and reach the genomic DNA, resulting in recombinase activity.

[0145] In some embodiments, expression of the at least one viral protein is controlled by light. In some embodiments, the expression level of the at least one viral protein is controlled byWSGR Docket No. 60885-743.601controlling one or more light parameters. In some embodiments, the light parameters comprise wavelength, intensity, exposure time or any combination thereof.

[0146] In some embodiments, expression of the at least one viral protein is controlled by using a closed loop control system. In some embodiments, expression of the at least one viral protein is controlled by using a closed loop control system comprising measuring the expression of the at least one viral protein, and controlling the expression of the at least one viral protein using light by adjusting the light parameters.

[0147] In some embodiments, the cell described herein comprises a mammalian cell. In some embodiments, the cell described herein comprises a human cell. In some embodiments, the cell described herein comprises a Human Embryonic Kidney 293 (HEK293) cell. In some embodiments, the cell described herein comprises a HEK293 cell, wherein the HEK293 cell does not comprise an adenoviral El gene. In some embodiments, the cell described herein comprises a Human Embryonic Kidney 293 T (HEK293T) cell. In some embodiments, the cell described herein comprises a HEK293T cell, wherein the HEK293T cell does not comprise an adenoviral El gene. In some embodiments, the cell described herein comprises a HeLa cell. In some embodiments, the cell described herein comprises a Chinese Hamster Ovary (CHO) cell. In some embodiments, the cell described herein comprises an insect cell. In some embodiments, the cell described herein comprises an Sf9 cell. In some embodiments, the cell described herein comprises a bacterial cell.

[0148] In some embodiments, the at least one exogenous nucleic acid described herein comprises a nucleotide sequence encoding a viral genome. In some embodiments, the viral genome described herein comprises a lentiviral genome. In some embodiments, the lentiviral genome comprises a long terminal repeat nucleic acid sequence (LTR). In some embodiments, the lentiviral genome comprises a 3’ LTR. In some embodiments, the lentiviral genome comprises a 5 ’LTR. In some embodiments, the lentiviral genome comprises the 3 ’LTR and the 5’ LTR.

[0149] In some embodiments, the lentiviral genome described herein is up to about 0.1 kilobases to about 15 kilobases. In some embodiments, the lentiviral genome is up to about 0.1 kilobases to about 0.5 kilobases, about 0.1 kilobases to about 1 kilobase, about 0.1 kilobases to about 3 kilobases, about 0.1 kilobases to about 5 kilobases, about 0.1 kilobases to about 7 kilobases, about 0.1 kilobases to about 9 kilobases, about 0.1 kilobases to about 12 kilobases, about 0.1 kilobases to about 15 kilobases, about 0.5 kilobases to about 1 kilobase, about 0.5 kilobases to about 3 kilobases, about 0.5 kilobases to about 5 kilobases, about 0.5 kilobases to about 7 kilobases, about 0.5 kilobases to about 9 kilobases, about 0.5 kilobases to about 12 kilobases, about 0.5 kilobases to about 15 kilobases, about 1 kilobase to about 3 kilobases, about 1 kilobase to about 5 kilobases, about 1 kilobase to about 7 kilobases, about 1 kilobase to about 9WSGR Docket No. 60885-743.601kilobases, about 1 kilobase to about 12 kilobases, about 1 kilobase to about 15 kilobases, about 3 kilobases to about 5 kilobases, about 3 kilobases to about 7 kilobases, about 3 kilobases to about 9 kilobases, about 3 kilobases to about 12 kilobases, about 3 kilobases to about 15 kilobases, about 5 kilobases to about 7 kilobases, about 5 kilobases to about 9 kilobases, about 5 kilobases to about 12 kilobases, about 5 kilobases to about 15 kilobases, about 7 kilobases to about 9 kilobases, about 7 kilobases to about 12 kilobases, about 7 kilobases to about 15 kilobases, about 9 kilobases to about 12 kilobases, about 9 kilobases to about 15 kilobases, or about 12 kilobases to about 15 kilobases long.

[0150] In some embodiments, the lentiviral genome described herein is up to about 0.1 kilobases, about 0.5 kilobases, about 1 kilobase, about 3 kilobases, about 5 kilobases, about 7 kilobases, about 9 kilobases, about 12 kilobases, or about 15 kilobases. In some embodiments, the lentiviral genome is up to at least about 0.1 kilobases, about 0.5 kilobases, about 1 kilobase, about 3 kilobases, about 5 kilobases, about 7 kilobases, about 9 kilobases, or about 12 kilobases. In some embodiments, the lentiviral genome is up to at most about 0.5 kilobases, about 1 kilobase, about 3 kilobases, about 5 kilobases, about 7 kilobases, about 9 kilobases, about 12 kilobases, or about 15 kilobases long.

[0151] In some embodiments, the viral genome described herein comprises an rAAV genome. In some embodiments, the rAAV genome comprises an inverted terminal repeat nucleic acid sequence (ITR). In some embodiments, the rAAV genome comprises a 3’ ITR. In some embodiments, the rAAV genome comprises a 5’ ITR. In some embodiments, the rAAV genome comprises the 5’ ITR and the 3’ ITR. In some embodiments, the rAAV genome comprises a mutant ITR that is not located at its 3’ or 5’ end. In some embodiments, an rAAV genome comprises the 3’ ITR, the 5’ ITR and the mutant ITR. In some embodiments, the rAAV genome is a self-complementary genome.

[0152] In some embodiments, the rAAV genome described herein is up to about 0.1 kilobases to about 7 kilobases. In some embodiments, the rAAV genome is up to about 0.1 kilobases to about 0.5 kilobases, about 0.1 kilobases to about 1 kilobase, about 0.1 kilobases to about 2 kilobases, about 0.1 kilobases to about 3 kilobases, about 0.1 kilobases to about 4 kilobases, about 0.1 kilobases to about 5 kilobases, about 0.1 kilobases to about 6 kilobases, about 0.1 kilobases to about 7 kilobases, about 0.5 kilobases to about 1 kilobase, about 0.5 kilobases to about 2 kilobases, about 0.5 kilobases to about 3 kilobases, about 0.5 kilobases to about 4 kilobases, about 0.5 kilobases to about 5 kilobases, about 0.5 kilobases to about 6 kilobases, about 0.5 kilobases to about 7 kilobases, about 1 kilobase to about 2 kilobases, about 1 kilobase to about 3 kilobases, about 1 kilobase to about 4 kilobases, about 1 kilobase to about 5 kilobases, about 1 kilobase to about 6 kilobases, about 1 kilobase to about 7 kilobases, about 2 kilobases to about 3 kilobases,WSGR Docket No. 60885-743.601about 2 kilobases to about 4 kilobases, about 2 kilobases to about 5 kilobases, about 2 kilobases to about 6 kilobases, about 2 kilobases to about 7 kilobases, about 3 kilobases to about 4 kilobases, about 3 kilobases to about 5 kilobases, about 3 kilobases to about 6 kilobases, about 3 kilobases to about 7 kilobases, about 4 kilobases to about 5 kilobases, about 4 kilobases to about 6 kilobases, about 4 kilobases to about 7 kilobases, about 5 kilobases to about 6 kilobases, about 5 kilobases to about 7 kilobases, or about 6 kilobases to about 7 kilobases. In some embodiments, the rAAV genome is up to about 0.1 kilobases, about 0.5 kilobases, about 1 kilobase, about 2 kilobases, about 3 kilobases, about 4 kilobases, about 5 kilobases, about 6 kilobases, or about 7 kilobases long.

[0153] In some embodiments, the rAAV genome described herein is up to at least about 0.1 kilobases, about 0.5 kilobases, about 1 kilobase, about 2 kilobases, about 3 kilobases, about 4 kilobases, about 5 kilobases, or about 6 kilobases. In some embodiments, the rAAV genome is up to at most about 0.5 kilobases, about 1 kilobase, about 2 kilobases, about 3 kilobases, about 4 kilobases, about 5 kilobases, about 6 kilobases, or about 7 kilobases long.

[0154] In some embodiments, the viral genome described herein comprises a gene expression cassette. As used herein, “gene expression cassette” means a recombinant nucleic acid construct comprising one or more nucleic acids described herein, wherein the recombinant nucleic acid construct is operably associated with at least one control sequence (e.g., a promoter).

[0155] In some embodiments, the viral genome described herein comprises a gene expression cassette, wherein the gene expression cassette comprises a transgene. As used herein, “transgene” means it is an exogenous nucleic acid encoding a polypeptide of interest.

[0156] In some embodiments, the gene expression cassette described herein comprises a promoter. In some embodiments, the promoter comprises an inducible promoter. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the promoter comprises a tissue-specific promoter. A promoter is tissue-specific when it only drives expression of at least one operably linked gene in specific tissues. In some embodiments, the promoter comprises a cell-type specific promoter. A promoter is cell specific when it only drives expression of at least one operably linked gene in specific cell types.

[0157] In some embodiments, the gene expression cassette described herein comprises a nucleic acid sequence encoding a payload. In some embodiments, the payload described herein comprises a therapeutic payload. As used herein, “therapeutic payload” means a payload that can be used to treat a disease. In some embodiments, the disease comprises a genetic disease. In some embodiments, the genetic disease comprises spinal muscular atrophy, Duchenne muscular dystrophy, hemophilia A and B, Leber congenital amaurosis, retinitis pigmentosa, genetic formsWSGR Docket No. 60885-743.601of Parkinson’s disease, Pompe disease, Friedreich’s ataxia, Batten disease, and amyotrophic lateral sclerosis.

[0158] In some embodiments, the payload described herein comprises a polypeptide. In some embodiments, the polypeptide comprises up to about 1 amino acid to about 3,000 amino acids. In some embodiments, the polypeptide comprises up to about 1 amino acid to about 10 amino acids, about 1 amino acid to about 50 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 500 amino acids, about 1 amino acid to about 1,000 amino acids, about 1 amino acid to about 1,500 amino acids, about 1 amino acid to about 2,000 amino acids, about 1 amino acid to about 2,500 amino acids, about 1 amino acid to about 3,000 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 1,000 amino acids, about 10 amino acids to about 1,500 amino acids, about 10 amino acids to about 2,000 amino acids, about 10 amino acids to about 2,500 amino acids, about 10 amino acids to about 3,000 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 1,000 amino acids, about 50 amino acids to about 1,500 amino acids, about 50 amino acids to about 2,000 amino acids, about 50 amino acids to about 2,500 amino acids, about 50 amino acids to about 3,000 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 1,000 amino acids, about 100 amino acids to about 1,500 amino acids, about 100 amino acids to about 2,000 amino acids, about 100 amino acids to about 2,500 amino acids, about 100 amino acids to about 3,000 amino acids, about 500 amino acids to about 1,000 amino acids, about 500 amino acids to about 1,500 amino acids, about 500 amino acids to about 2,000 amino acids, about 500 amino acids to about 2,500 amino acids, about 500 amino acids to about 3,000 amino acids, about 1,000 amino acids to about 1,500 amino acids, about 1,000 amino acids to about 2,000 amino acids, about 1,000 amino acids to about 2,500 amino acids, about 1,000 amino acids to about 3,000 amino acids, about 1,500 amino acids to about 2,000 amino acids, about 1,500 amino acids to about 2,500 amino acids, about 1,500 amino acids to about 3,000 amino acids, about 2,000 amino acids to about 2,500 amino acids, about 2,000 amino acids to about 3,000 amino acids, or about 2,500 amino acids to about 3,000 amino acids. In some embodiments, the polypeptide comprises up to about 1 amino acid, about 10 amino acids, about 50 amino acids, about 100 amino acids, about 500 amino acids, about 1,000 amino acids, about 1,500 amino acids, about 2,000 amino acids, about 2,500 amino acids, or about 3,000 amino acids. In some embodiments, the polypeptide comprises up to at least about 1 amino acid, about 10 amino acids, about 50 amino acids, about 100 amino acids, about 500 amino acids, about 1,000 amino acids, about 1,500 amino acids, about 2,000 amino acids, or about 2,500 amino acids.WSGR Docket No. 60885-743.601

[0159] In some embodiments, the polypeptide described herein comprises up to at most about 10 amino acids, about 50 amino acids, about 100 amino acids, about 500 amino acids, about 1,000 amino acids, about 1,500 amino acids, about 2,000 amino acids, about 2,500 amino acids, or about 3,000 amino acids.

[0160] In some embodiments, the payload comprises an RNA molecule. In some embodiments, the RNA molecule described herein is up to about 0.1 kilobases to about 15 kilobases. In some embodiments, the RNA molecules up to about 0.1 kilobases to about 0.5 kilobases, about 0.1 kilobases to about 1 kilobase, about 0.1 kilobases to about 3 kilobases, about 0.1 kilobases to about 5 kilobases, about 0.1 kilobases to about 7 kilobases, about 0.1 kilobases to about 9 kilobases, about 0.1 kilobases to about 12 kilobases, about 0.1 kilobases to about 15 kilobases, about 0.5 kilobases to about 1 kilobase, about 0.5 kilobases to about 3 kilobases, about 0.5 kilobases to about 5 kilobases, about 0.5 kilobases to about 7 kilobases, about 0.5 kilobases to about 9 kilobases, about 0.5 kilobases to about 12 kilobases, about 0.5 kilobases to about 15 kilobases, about 1 kilobase to about 3 kilobases, about 1 kilobase to about 5 kilobases, about 1 kilobase to about 7 kilobases, about 1 kilobase to about 9 kilobases, about 1 kilobase to about 12 kilobases, about 1 kilobase to about 15 kilobases, about 3 kilobases to about 5 kilobases, about 3 kilobases to about 7 kilobases, about 3 kilobases to about 9 kilobases, about 3 kilobases to about 12 kilobases, about 3 kilobases to about 15 kilobases, about 5 kilobases to about 7 kilobases, about 5 kilobases to about 9 kilobases, about 5 kilobases to about 12 kilobases, about 5 kilobases to about 15 kilobases, about 7 kilobases to about 9 kilobases, about 7 kilobases to about 12 kilobases, about 7 kilobases to about 15 kilobases, about 9 kilobases to about 12 kilobases, about 9 kilobases to about 15 kilobases, or about 12 kilobases to about 15 kilobases long.

[0161] In some embodiments, the RNA molecule described herein is up to about 0.1 kilobases, about 0.5 kilobases, about 1 kilobase, about 3 kilobases, about 5 kilobases, about 7 kilobases, about 9 kilobases, about 12 kilobases, or about 15 kilobases. In some embodiments, the RNA molecule is up to at least about 0.1 kilobases, about 0.5 kilobases, about 1 kilobase, about 3 kilobases, about 5 kilobases, about 7 kilobases, about 9 kilobases, or about 12 kilobases. In some embodiments, the RNA molecule is up to at most about 0.5 kilobases, about 1 kilobase, about 3 kilobases, about 5 kilobases, about 7 kilobases, about 9 kilobases, about 12 kilobases, or about 15 kilobases long.

[0162] In some embodiments, the payload comprises an RNA molecule and a polypeptide molecule. In some embodiments, the payload comprises an RNA-protein complex.

[0163] In some embodiments, the cell described herein produces a viral particle. In some embodiments, the viral particle described comprises a lentiviral particle. In some embodiments, the viral particle described comprises an rAAV particle. In some embodiments, the rAAV particleWSGR Docket No. 60885-743.601described herein comprises an rAAVl particle, an rAAV2 particle, an rAAV3 particle, an rAAV4 particle, an rAAV5 particle, an rAAV6 particle, an rAAV7 particle, an rAAV8 particle, an rAAV9 particle, an rAAVIO particle, an rAAVl 1 particle, an rAAV12 particle, an rAAV13 particle, any variant thereof, or any combination thereof.

[0164] As described herein, “empty rAAV particle” means an rAAV particle that does not comprise an rAAV genome.

[0165] In some embodiments, the cell described herein produces a plurality of rAAV particles, wherein the percentage of empty rAAV particles is fewer than about 0.1 % to about 95 %. In some embodiments, the cell described herein produces a plurality of rAAV particles, wherein the percentage of empty rAAV particles is fewer than about 0.1 % to about 1 %, about 0.1 % to about 10 %, about 0.1 % to about 20 %, about 0.1 % to about 30 %, about 0.1 % to about 40 %, about 0.1 % to about 50 %, about 0.1 % to about 60 %, about 0.1 % to about 70 %, about 0.1 % to about 80 %, about 0.1 % to about 90 %, about 0.1 % to about 95 %, about 1 % to about 10 %, about 1 % to about 20 %, about 1 % to about 30 %, about 1 % to about 40 %, about 1 % to about 50 %, about 1 % to about 60 %, about 1 % to about 70 %, about 1 % to about 80 %, about 1 % to about 90 %, about 1 % to about 95 %, about 10 % to about 20 %, about 10 % to about 30 %, about 10 % to about 40 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 10 % to about 95 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 20 % to about 95 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 30 % to about 95 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 40 % to about 95 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 50 % to about 95 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 60 % to about 95 %, about 70 % to about 80 %, about 70 % to about 90 %, about 70 % to about 95 %, about 80 % to about 90 %, about 80 % to about 95 %, or about 90 % to about 95 %. In some embodiments, the cell described herein produces a plurality of rAAV particles, wherein the percentage of empty rAAV particles is fewer than about 0.1 %, about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, or about 95 %. In some embodiments, the cell described herein produces a plurality of rAAV particles, wherein the percentage of empty rAAV particles is fewer than at least about 0.1 %, about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, or about 90 %. In some embodiments, the cell describedWSGR Docket No. 60885-743.601herein produces a plurality of rAAV particles, wherein the percentage of empty rAAV particles is fewer than at most about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, or about 95 %.

[0166] In some embodiments, the cell described herein comprises a packaging cell. As described herein, “packaging cell” means a cell that can produce viral particles upon transfection with a plasmid encoding the viral genome combined with chemical induction, illumination, removal of light or any combination thereof. The packaging cell described herein can be generated by introducing at least one exogenous nucleic acid described herein into a cell described herein using a vector described herein. In some embodiments, the packaging cell described herein comprises at least one exogenous nucleic acid comprising, the at least one promoter described herein, the first nucleic acid sequence encoding the at least one viral protein described herein, the second nucleic acid sequence encoding the light controllable transcriptional regulator described herein, a nucleic acid sequence encoding the adenovirus VA RNA described herein, and a nucleic acid sequence encoding the AAV Rep, Cap and helper proteins described herein that are not under inducible control, or any combination thereof. In some embodiments, the at least one viral protein described herein comprises an AAV Rep protein described herein and an AAV Capsid protein (e.g., VP1, VP2, or VP3) described herein. In some embodiments, the AAV Rep protein described herein comprises an AAV Rep78 protein described herein, an AAV Rep68 protein described herein, an AAV Rep52 protein described herein, an AAV Rep40 protein described herein or any combination thereof. In some embodiments, the AAV Capsid protein described herein comprises the AAV VP1 protein described herein, the AAV VP2 protein described herein, the AAV VP3 protein described herein, or any combination thereof.

[0167] In some embodiments, the cell described herein comprises a producer cell. As described herein, “producer cell” means a cell that can produce viral particles. In some embodiments, the producer cell produces viral particles constitutively. In some embodiments, the producer cell produces viral particles upon chemical induction, illumination, removal of light or any combination thereof. In some embodiments, the producer cell can produce viral particles described herein. The producer cell described herein can be generated by adding a nucleic acid encoding the viral genome into the packaging cells described herein. In some embodiments, the viral genome described herein comprises the lentiviral genome described herein. In some embodiments, the viral genome described herein comprises the rAAV genome described herein.

[0168] As illustrated in FIG. 8, in some embodiments, an exogeneous nucleic acid comprising at least one optogenetic (opto) transactivator is stably integrated into Viral Production Cells 2.0 (VPC2) cells, generating an optogenetic chassis (OptoChassis) cell line following selection and / or clonal expansion. The OptoChassis cell line comprises at least one optogeneticWSGR Docket No. 60885-743.601transactivator under the control of at least one non-optogenetic promoter. In some embodiments, at least one replication protein under the control of at least one optogenetic promoter (OptoPromoter) and at least one helper protein under the control of at least one optogenetic promoter (OptoPromoter) are stably integrated into the OptoChassis cell line, thereby generating a packaging cell line after selection and / or clonal expansion. In this case, the packaging cell line comprises: (i) at least one optogenetic transactivator under the control of a promoter; (ii) at least one replication protein under OptoPromoter control; and (iii) at least one helper protein under OptoPromoter control. In some embodiments, the OptoPromoter controlling the at least one replication protein is different than the OptoPromoter controlling the at least one helper protein.

[0169] In some embodiments, the packaging cell line is transfected with a vector (e.g., pAAV-CapGOI, as shown in Fig. 8) comprising at least one gene expression cassette and at least one capsid protein. The gene expression cassette may comprise at least one nucleic acid sequence encoding at least one payload or gene of interest (GOI) under the control of at least one promoter. In some embodiments, the at least one promoter for the gene expression cassette is a non-optogenetic promoter. In some embodiments, the at least one capsid protein in the vector (e.g., pAAV-CapGOI, as shown in Fig. 8) may be under the control of at least one promoter. In some embodiments, the at least one promoter for the at least one capsid protein is a non-optogenetic promoter. In this case, following the plasmid transfection of the packaging cell line and light-controllable expression, viral particles (e.g., AAVs) can be produced.

[0170] In some embodiments, the vector (e.g., pAAV-CapGOI, as shown in Fig. 8) comprising at least one gene expression cassette and at least one capsid protein can be stably integrated into the packaging cell line, generating a fully inducible producer cell line following clonal expansion. The gene expression cassette may comprise a nucleic acid sequence encoding at least one payload or gene of interest (GOI) under the control of at least one promoter. In some embodiments, the at least one promoter for the gene expression cassette is a non-optogenetic promoter. In some embodiments, the at least one capsid protein in the vector (e.g., pAAV-CapGOI, as shown in Fig. 8) may be under the control of at least one promoter. In some embodiments, the at least one promoter for the at least one capsid protein is a non-optogenetic promoter. In this configuration, viral particles (e.g., AAVs) can be produced following light-controllable expression using the fully inducible producer cell line. These examples are illustrative only; the order, configuration, and position of the helper proteins, replication proteins, capsid proteins, or gene of interest (GOI) may be altered.

[0171] Provided herein is a cell culture comprising a plurality of the cells described herein and a cell culture media. In some embodiments, the plurality of the cells is in suspension.WSGR Docket No. 60885-743.601

[0172] Provided herein is a bioreactor comprising the cell culture described herein. In some embodiments, the bioreactor comprises the suspension cell culture described herein. The bioreactor may be any type of culture vessel suitable for growing cells in a suspension culture. In some embodiments, the bioreactor vessel comprises a total volume of at least about 0.05L, at least about 0. IL, at least about 0.2L, at least about 0.3L, at least about 0.4L at least about 0.5L, at least about 0.6L, at least about 0.7L, at least about 0.8L, at least about 0.9L, at least about IL, at least about 5L, at least about 10L, at least about 50L, at least about 100 L, at least about 150 L, at least about 200 L, at least about 250 L, at least about 300 L, at least about 400 L, at least about 500 L, at least about 600 L, at least about 700 L, at least about 750 L, at least about 800 L, at least about 900 L, at least about 1000 L, at least about 2000 L, at least about 3000 L, at least about 5000 L, or more.

[0173] In some embodiments, the bioreactor comprises one or more light sources, wherein the one or more light source comprises light emitting diodes (LEDs). In some embodiments, the one or more LEDs comprises at least two different LEDs. In some embodiments, the one or more LEDs comprises at least two different LEDs, wherein the at least two different LEDs emit light at different wavelengths. In some embodiments, the bioreactor comprises one or more light sources, wherein the one or more light source comprises one or more lasers. In some embodiments, the bioreactor comprises one or more light sources, wherein the one or more light source comprises an incandescent light source.

[0174] In some embodiments, the bioreactor comprises one or more light sources, wherein the one or more light source is located inside the bioreactor, or located on an interior surface of the bioreactor.

[0175] In some embodiments, the bioreactor comprises one or more light sources, wherein the one or more light source is located outside the bioreactor, or on an exterior surface of the bioreactor.

[0176] In some embodiments, the bioreactor comprises at least one wall or surface that is optically transparent.

[0177] In some embodiments, the bioreactor comprises a temperature source for controlling a temperature of the culture media.

[0178] In some embodiments, the bioreactor comprises an agitation source for agitating the culture media.

[0179] In some embodiments, the bioreactor comprises a controller for controlling illumination.II. Light-Controllable Transcriptional Regulators and Associated ComponentsWSGR Docket No. 60885-743.601

[0180] Provided herein are additional or alternative details associated with the light-controllable transcriptional regulators.

[0181] In some embodiments, a promoter (that is operably linked to a polynucleotide sequence) may comprise a constitutive promoter that provides continuous transcription activity in a manner independent of external regulatory inputs. The promoter unit may comprise a synthetic promoter. In some cases, the promoter unit comprises a synthetic minimal promoter. In some cases, the promoter unit comprises a synthetic full promoter. In some cases, the promoter unit may comprise (i) a promoter region that provides a basal level of transcriptional activity (e.g., a minimal promoter) and (ii) a DNA-binding recognition element positioned upstream, downstream, or within the promoter (e.g., such that, upon light exposure, the light-controllable transcriptional regulator may be activated to bind to the DNA binding recognition element or may be activated to be removed from the DNA binding recognition element). Table 3 provides nonlimiting examples of constitutive promoters and minimal promoters.Table 3. Non-limiting examples of promoters

[0182] In some embodiments, the DNA-binding recognition elements can be configured to specifically associate (e.g., directly or indirectly) with a corresponding DNA-binding domain of the light-controllable transcriptional regulator. Table 4 provides nonlimiting exemplary DNA-binding recognition elements and its cognate DNA binding domain.WSGR Docket No. 60885-743.601Table 4. Non-limiting exemplary DNA-recognition sequence and corresponding DNA-binding domains

[0183] In some cases, the DNA-binding recognition elements can be configured to specifically associate with a corresponding DNA-binding domain of a regulatory factor (e.g., a light-controllable transcriptional regulator). In some cases, the promoter unit may respond to a light comprising a wavelength or wavelength range (e.g., an optogenetic promoter unit). In some embodiments, the optogenetic promoter unit can refer to a promoter unit that drives transcription in a light-dependent manner. In some cases, a regulatory factor that interacts with the optogenetic promoter unit (e.g., at the DNA binding recognition element) can be a light-controllable transcriptional regulator.

[0184] In some cases, the light-controllable transcriptional regulator can comprise a cognate DNA-binding domain coupled to (e.g., directly or indirectly) a light-responsive protein (e.g., comprising LOV, CRY2, or PhyB domains) such that illumination, changes in one or more illumination parameters, or removal of light may induce recruitment, dissociation, conformation change, or complex formation enabling binding or dissociation of the regulatory factor to / from the DNA-binding recognition element. Upon such association or dissociation of the regulatory factor from the promoter unit (e.g., at the DNA-binding recognition element), the promoter unitWSGR Docket No. 60885-743.601(e.g., the optogenetic promoter unit) can initiate or terminate transcription of the operably linked nucleic acid sequence in a light-dependent manner. In some cases, the light-controllable transcriptional regulator may comprise a transcriptional activator domain (e.g., VP 16 or VPR) or a transcriptional repressor domain (KRAB). In some embodiments, the transcriptional activator domain can comprise VP 16, VP64, VPR, p65, Rta, or a combination thereof. In some embodiments, the transcription suppressor domain can be KRAB, SID, REST / NTSF, or a combination thereof.

[0185] In some embodiments, a light-controllable transcriptional regulator may be a transcriptional activator fused to (e.g., directly or indirectly) a light-controllable protein. In some embodiments, a light-controllable transcriptional regulator of the one or more light-controllable transcriptional regulators may be a transcriptional suppressor fused to (e.g., directly or indirectly) a light-controllable protein. In some cases, a light-controllable protein can be homodimer, oligomer, and / or heterodimer.

[0186] For example, in some cases, a light-controllable transcriptional regular may comprise, from N-terminus to C-terminus:[DNA-binding domain]-[light-controllable protein]-[transcriptional activator];[DNA-binding domain]-[transcriptional activator]-[light-controllable protein];[light-controllable protein]-[transcriptional activator]-[DNA-binding domain];[light-controllable protein]-[DNA-binding domain]-[transcriptional activator];[transcriptional activator] -[DNA-binding domain]-[light-controllable protein]; or [transcriptional activator] -[light-controllable protein]-[DNA-binding domain], wherein indicates an optional linker. In some cases, in the presence or absence of light, the light-controllable proteins (e.g., homodimer) can dimerize.

[0187] In some cases, a light-controllable transcriptional regular may comprise, from N-terminus to C-terminus:[DNA-binding domain]-[light-controllable protein 1] and [light-controllable protein 2] - [transcriptional activator];[light-controllable protein l]-[DNA-binding domain] and [light-controllable protein 2] - [transcriptional activator];[DNA-binding domain]-[light-controllable protein 1] and [transcriptional activator] - [light-controllable protein 2]; or[DNA-binding domain]-[light-controllable protein 1] and [light-controllable protein 2] - [transcriptional activator],wherein indicates an optional linker. In some cases, in the presence or absence of light, the light-controllable protein 1 and the light-controllable protein 2 (e.g., heterodimer) can dimerize.WSGR Docket No. 60885-743.601

[0188] The methods, cells, cell cultures, and compositions described herein may comprise a light-controllable protein. In some cases, the light-controllable protein may be an optogenetic switch. In some embodiments, the optogenetic switch is a dimerization-based optogenetic switch. In some embodiments, the dimerization-based optogenetic switch is a heterodimerization-based optogenetic switch, as described herein. In some embodiments, the dimerization-based optogenetic switch is a homodimerization-based optogenetic switch, as described herein. Any light-controllable protein may be used, including any light-controllable protein described herein. Non-limiting examples of optogenetic dimerization systems suitable for use with the methods and systems provided herein are described in Table 2.

[0189] In various aspects, the light-controllable protein can comprise EL222, Light-Oxygen-Voltage (LOV) Domain, PhyB-PIF, PhyA-PIF, PhyA-FHYl, PhyA-FHL, UVR8-COP1, VVD, Blue-light-using FAD (BLUF) photoreceptor domain, Dronpa, Dronpal45N, Deinococcus radiodurans bacterial phytochrome (DrBphP), MagRed, NanoRed, Cobalamin binding domains (CBD), CRY2 / CIB1 unit, Opto-Cas, Opti-dCas, BphPl-QPASl, BphS / BphO unit, Cphl, Cph8, or OptoXRs.

[0190] In some embodiments, a light-controllable protein described herein is a hybrid system. For example, in some embodiments, the methods and compositions described herein comprise a p65-Gal4-LOV (eGAV, such as eGAV#12) transcription factor or a variant thereof. The eGAV inducer is a hybrid protein comprising an activation domain of the transcription factor p-65 from HEK293 cells; a DNA binding domain of the Gal4 transcription factor; and a LOV domain of the Vivid (VVD) protein from the species Neurospora crassa, which responds to blue light. In response to blue light, two VVD domains form a homodimer that results in dimerization of the Gal4 domains. In this homodimer state, Gal4 recognizes the Gal4 activation sequence (UAS) fused to a minimal basal promoter, consequently recruiting the entire transcription machinery via the p65 domain and then activating transcription of a gene of interest. In some embodiments, the eGAV protein as provided herein may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more amino acid mutations (e.g., deletion, substitution, or addition) as compared to SEQ ID NO: 1 (see Table 5). In some embodiments, the eGAV constructs provided herein may have an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or substantially 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the eGAV constructs provided herein may have an amino acid sequence having at most about 99%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, atWSGR Docket No. 60885-743.601most about 75%, or at most about 70% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0191] Amino acid sequences of example polypeptide constructs for the methods, cells, cell cultures, and bioreactors for producing viral protein(s) are provided in Table 5. The example polypeptide constructs include an example light-controllable transcriptional regulator (e.g., eGAV) and viral proteins, including viral Rep proteins (e.g., Rep40, Rep52, Rep68, Rep68 with M225G mutation to ablate the native start codon of Rep40 within Rep68 thereby inactivating expression of Rep40), viral helper proteins (e.g., 2EA, L4 (22k), L4 (33k), E4orf6, E4orf6 / 7), and viral capsid proteins (e.g., VP1, VP2, VP3).

[0192] In some embodiments, a viral protein provided herein may have an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or substantially 100% sequence identity to any one of the amino acid sequence set forth in SEQ ID NOs: 2-15. In some embodiments, the viral protein provided herein may have an amino acid sequence having at most about 99%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, or at most about 70% sequence identity to any one of the amino acid sequence set forth in SEQ ID NOs: 2-15. In some embodiments, nucleic acid sequences (e.g., vectors) provided herein may encode any one of such viral protein.

[0193] Example nucleic acid sequences encoding one or more viral proteins (e.g., from start codon to final stop codon) for the methods, cells, cell cultures, and bioreactors for producing viral protein(s) are provided in Table 6. Such viral protein(s) can include, for example, a combination of E2A, E4, and L4 (e.g., for plasmid P 2405); a combination of E2A and L4 (e.g., for plasmid P 2414); a combination of E2A, L4, and E4 (e.g., P 2415 or P_2420); a combination of Rep40 and Rep68 having M225G mutation (e.g., P 2402); a combination of Rep52 and Rep78 having M225G mutation (e.g., P_2427), a combination of Rep52 and Rep78 without M225G mutation (e.g., P 2428); Rep40 wherein the nucleic acid sequence encoding the Rep40 comprises a first codon optimization (codon optimization 1 or “Codl”) (e.g., P 2436); Rep40 wherein the nucleic acid sequence encoding the Rep40 comprises a second codon optimization (codon optimization 2 or “Cod2”) (e.g., P_2437); Rep52 wherein the nucleic acid sequence encoding the Rep52 comprises “Codl” (e.g., P 2438); Rep52 wherein the nucleic acid sequence encoding the Rep52 comprises “Cod2” (e.g., P 2439); Rep68 wherein the nucleic acid sequence encoding the Rep68 comprises “Codl” (e.g., P 2440); Rep68 wherein the nucleic acid sequence encoding the Rep68 comprises “Cod2” (e.g., P 2441); Rep78 wherein the nucleic acid sequence encoding the Rep78 comprises “Codl” (e.g., P 2442); Rep78 wherein the nucleic acid sequence encoding the Rep78WSGR Docket No. 60885-743.601comprises “Cod2” (e.g., P_2443); and a combination of Rep52 with Codl and Rep78 with Cod2 (e.g., P 2444). In some cases, all Rep68 and Rep78 in the constructed abovementioned may also contain the M225G mutation).

[0194] In some embodiments, a viral protein provided herein may be encoded by a polynucleotide molecule comprising a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or substantially 100% sequence identity to any one of the nucleic acid sequence set forth in SEQ ID NOs: 21-36. In some embodiments, a viral protein provided herein may be encoded by a polynucleotide molecule comprising a polynucleotide sequence having at most about 99%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, or at most about 70% sequence identity to any one of the amino acid sequence set forth in SEQ ID NOs: 21-36.Table 5. Example polypeptide sequences.WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601Table 6. Example nucleic acid sequences encoding one or more viral proteinsWSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601WSGR Docket No. 60885-743.601III. Additional Details

[0195] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example reference to “the cell” includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included.WSGR Docket No. 60885-743.601

[0196] As used herein, in any instance or embodiment described herein, “comprising” may be replaced with “consisting essentially of’ and / or “consisting of,” unless context clearly connotes otherwise. Similarly, as used herein, in any instance or embodiment described herein, “comprises” may be replaced with “consists essentially of’ and / or “consists of,” unless context clearly connotes otherwise.

[0197] As used herein, the term “about” in connection with a number refers to that number plus or minus 10%. When used in connection with a range, the term “about” refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0198] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each were set out individually herein.

[0199] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0200] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0201] The “percent sequence identity” between a reference amino acid sequence and a query amino sequence (i.e., the amino sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference amino acid sequence) is determined by aligning the sequences using the Needleman-Wunsch alignment algorithm as implemented using the “Global Align” BLAST program available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi (with a gap existence penalty of 11 and a gap extension penalty of 1) and comparing the sequences. The number of exact matches, divided by the total number of positions in the alignment (which corresponds with the number of amino acids in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query amino acid sequence and the reference amino acid sequence (i.e., percent sequence identity = (# of exact matches / (total # of positions in alignment)* 100). The “percent sequence identity” between a reference nucleic acid sequence and a query nucleic acid sequence (i.e., the nucleic acid sequence being analyzed toWSGR Docket No. 60885-743.601determine whether it is within a particular percent sequence identity with the reference nucleic acid sequence) is determined by aligning the sequences using the Needleman-Wunsch alignment algorithm as implemented using the “Global Align” BLAST program available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi (with match / mismatch scores of 2,-3, a gap existence penalty of 5, and a gap extension penalty of 2) and comparing the aligned nucleic acids. The number of exact matches divided by the total number of nucleotides in the alignment (which corresponds with the number of nucleotides in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query nucleic acid sequence and the reference nucleic acid sequence (i.e., percent sequence identity = (# of exact matches) / (total # of nucleotides in the alignment)* 100).EXAMPLESExample 1 - Producing rAAV8 particles using HEK293 cells stably engineered to produce AAV Rep proteins upon illumination

[0202] Briefly, rAAV8 particles are created by first generating cells comprising at least one gene expression cassette encoding AAV8 Rep proteins under the transcriptional control of a light inducible promoter. For production of viral vectors, the cells are transfected with three plasmids comprising an rAAV genome (e.g., expressing eGFP), AAV Capsid genes, and adenoviral helper genes, respectively, and expression of the AAV Rep proteins is induced upon illumination with the appropriate wavelength of light or removal of the appropriate wavelength of light.

[0203] The produced rAAV8 particles express eGFP under the transcriptional control of a ubiquitous CMV promoter. The rAAV8 particles generated by the methods described herein are used to further assess the transduction efficiency of rAAV8 in vitro.Generating a cell that expresses AAV8 Rep78, A A 1'8 Rep68, A A 1'8 Rep52 and / or A A 1'8 Rep40 protein when illuminated with appropriate wavelengths of light.

[0204] First, four transposon donor plasmids are generated via Gibson assembly of DNA molecules. The first transposon donor plasmid encodes a first light-controllable transcriptional regulator and an AAV8Nine Rep78 protein operably linked by a first light inducible promoter. The second transposon donor plasmid encodes a second light-controllable transcriptional regulator and an AAV8 Rep68 protein operably linked by a second light inducible promoter. The third transposon donor plasmid encodes a third light-controllable transcriptional regulator and an AAV8 Rep52 protein operably linked by a third light inducible promoter. The fourth transposonWSGR Docket No. 60885-743.601donor plasmid encodes a fourth light-controllable transcriptional regulator and an AAV8 Rep40 protein operably linked by a fourth light inducible promoter.

[0205] For engineering of the HEK293 cells, the cells are transfected with each of the four transposon donor plasmids and a transposase (e.g., encoded on a plasmid or mRNA) to allow stable integration.

[0206] The engineered HEK293 cells comprise 4 gene cassettes, each capable of expressing AAV8 Rep78, AAV8 Rep68, AAV8 Rep52 or AAV8 Rep40 protein by activating a corresponding light controllable transcriptional regulator upon illumination with different wavelengths of light. The engineered cell reversibly expresses each of the AAV8 Rep proteins upon illumination with the appropriate wavelengths of light or removal of the appropriate wavelengths of light.

[0207] Optionally, the HEK293 cells do not comprise an adenovirus El gene.

[0208] Optionally, the nucleotide sequences that encode the AAV8 Rep78 and AAV8 Rep68 are codon-optimized to be deficient of the internal pl9 promoter, preventing the AAV8 Rep52 and AAV8 Rep40 proteins from being expressed independent of induction.

[0209] Optionally, the pl9 promoter regions of the nucleotide sequences that encode AAV8 Rep78 and AAV8 Rep68 proteins are codon-optimized to be deficient of the internal pl9 promoter. The remaining sequence is natural to allow alternative mRNA splicing for AAV8 Rep78 / Rep68 and AAV8 Rep52 / Rep40 production.

[0210] Optionally, only a subset of the AAV8 Rep78, AAV8 Rep68, AAV8 Rep52 or AAV8 Rep40 is used.

[0211] Optionally, all four AAV Rep proteins are under the transcriptional control of the same light controllable transcriptional regulator. First, a transposon donor plasmid is generated via Gibson assembly of DNA molecules. The transposon donor plasmid encodes a light-controllable transcriptional regulator and a polycistronic gene that encodes a polypeptide comprising an AAV8 Rep78 protein, an AAV8 Rep68 protein, an AAV8 Rep 52 protein and an AAV8 Rep40 protein separated by self-cleaving T2A peptides, wherein expression of the polycistronic gene is operably linked by a light inducible promoter. Optionally, the transposon donor plasmid encodes Rep78 / 68 and Rep52 / 40 separated by a self-cleaving T2A peptide, wherein expression of the polycistronic gene is operably linked by a light inducible promoter. HEK293 cells are then transfected with the transposon donor plasmid and a transposase (e.g., encoded on a plasmid or mRNA) to allow stable integration. The engineered cell reversibly expresses all four AAV Rep proteins upon illumination with the appropriate wavelengths of light or removal of the appropriate wavelengths of light.WSGR Docket No. 60885-743.601

[0212] Optionally, the expression of AAV8 Rep78, AAV8 Rep68, AAV8 Rep52 and / or AAV8 Rep40 protein is controlled via a closed loop system. The closed loop system comprises measuring the expression of AAV8 Rep78, AAV8 Rep68, AAV8 Rep52 and / or AAV8 Rep40 protein, and adjusting their expression levels using light by adjusting light parameters, such as intensity, wavelength, or exposure time. The detection of the Rep protein expression is, for example, done using a destabilized fluorescent protein whose level is proportional to the Rep protein (e.g., via a week IRES: PLight-REP-IRES-FluorescentProtein, or a leaky stop codon: Pught-REP-STOP-FluorescentProtein). Alternatively, the level of Rep proteins is estimated by measuring the concentration of the viral genomes in the cells. Alternatively, Raman spectroscopy is used.Generating rAA V particles

[0213] The cells are then transfected with three additional plasmid constructs via chemical transfection (such as lipofectamine). The first plasmid construct comprises a nucleotide sequence encoding AAV8 VP1, AAV8 VP2 and AAV8 VP3 capsid proteins. The second plasmid construct comprises a nucleotide sequence that encodes adenovirus E4 protein, adenovirus E2A protein and adenovirus VA RNA. The third plasmid construct, AAV-CMV-EGFP, comprises a nucleotide sequence encoding an rAAV genome. The transfected cells are then stimulated with an appropriate wavelength of light to trigger expression of the AAV8 Rep proteins. The AAV8 Rep proteins trigger replication of the rAAV genome and assembly of rAAV8 particles.Collecting rAAV particles

[0214] The cell culture containing rAAV8 particles is collected 4 days post-onset of illumination with the appropriate wavelength of light. Both the cells (pelleted by centrifugation) and the cell supernatant are harvested.Purifying and concentrating rAAV particles

[0215] The rAAV particles are precipitated from the supernatant by adding polyethylene glycol (PEG), gently mixing the solution, and incubating the solution at 4°C. The solution is then centrifuged to generate a PEG pellet containing the rAAV particles. The cell pellet is resuspended in a high salt buffer comprising a salt-activated nuclease to lyse any intact cells and digest any DNA not encapsidated in an rAAV capsid. The PEG pellet is also resuspended in a high salt buffer comprising a salt-activated nuclease to lyse any intact cells and digest any DNA not encapsidated in an rAAV capsid. The resuspended PEG pellets and cell pellets are pooled. The pooled pellets are purified and concentrated using iodixanol gradient ultracentrifugation. The fraction containing the purified rAAV particles is collected, and any residual iodixanol is removed via buffer exchange using an Amicon filtration device.AAV titration via ddPCRWSGR Docket No. 60885-743.601

[0216] The purified and concentrated rAAV8 particles are titrated via ddPCR using primers complimentary to the AAV internal terminal repeat sequences.Determining the percentage of empty AA V particles

[0217] The percentage of empty rAAV8 particles is determined using Transmission electron microscopy (TEM). The empty capsid ratio of the purified and concentrated rAAV particles is less than 95%.Assessing rAA V transduction efficiency in vitro

[0218] HepG2 (human hepatocellular carcinoma cells), Huh7 (human hepatocellular carcinoma cells), C2C12 (mouse myoblast cells), and HEK293 cells are seeded at a density of 5,000 cells cm -2 in a 96-well plate. After 24 h, viral vectors at different MOI (1, 10, 100, 1,000, 10,000, 100,000, 1,000,000) are added and after another 48 h the transduction efficiency of the rAAV8 particles for the different cell lines is assessed by measuring the percentage of cells that express eGFP via flow cytometry.Example 2 - Producing rAAV particles using packaging cells

[0219] rAAV8 particles are created by first generating a packaging cell comprising adenoviral helper genes and at least two gene expression cassettes, the first encoding AAV8 Rep proteins and the second encoding AAV8 Capsid proteins, each under the transcriptional control of a corresponding light inducible promoter. For production of viral vectors, the cells are transfected with a plasmid comprising an rAAV genome and expression of the AAV Rep and Cap proteins is induced upon illumination with the appropriate wavelengths of light or removal of the appropriate wavelengths of light.

[0220] The produced rAAV8 particles express eGFP under the transcriptional control of a ubiquitous CMV promoter. The rAAV8 particles generated by the methods described herein are used to further assess the transduction efficiency of rAAV8 in vitro.Generating a packaging cell that expresses A A 1'8 Rep and / or AAV8 Capsid proteins when illuminated with appropriate wavelengths of light.

[0221] First, eight transposon donor plasmids are generated via Gibson assembly of DNA molecules. The first transposon donor plasmid encodes a first light-controllable transcriptional regulator and an AAV8 Rep78 protein operably linked by a first light inducible promoter. The second transposon donor plasmid encodes a second light-controllable transcriptional regulator and an AAV8 Rep68 protein operably linked by a second light inducible promoter. The third transposon donor plasmid encodes a third light-controllable transcriptional regulator and an AAV8 Rep52 protein operably linked by a third light inducible promoter. The fourth transposon donor plasmid encodes a fourth light-controllable transcriptional regulator and an AAV8 Rep40 protein operably linked by a fourth light inducible promoter. The fifth transposon donor plasmidWSGR Docket No. 60885-743.601encodes a fifth light-controllable transcriptional regulator and an AAV8 VP1 protein operably linked by a fifth light inducible promoter. The sixth transposon donor plasmid encodes a sixth light-controllable transcriptional regulator and an AAV8 VP2 protein operably linked by a sixth light inducible promoter. The seventh transposon donor plasmid encodes a seventh light-controllable transcriptional regulator and an AAV8 VP3 protein operably linked by a seventh light inducible promoter. The eighth transposon donor plasmid encodes the adenoviral helper genes encoding adenovirus E4 protein, adenovirus E2A protein, and adenovirus VA RNA.

[0222] For engineering of the HEK293 cells, the cells are transfected with each of the eight transposon donor plasmids and a transposase (e.g. encoded on a plasmid or mRNA) to allow stable integration.

[0223] The engineered HEK293 cells comprise 7 gene cassettes, each capable of expressing AAV8 Rep78, AAV8 Rep68, AAV8 Rep52, AAV8 Rep40, AAV8 VP1, AAV8 VP2 or AAV8 VP3 protein by activating a corresponding light controllable transcriptional regulator upon illumination with the appropriate wavelengths of light. The engineered cell reversibly expresses each of the AAV8 Rep and / or AAV8 Cap proteins upon illumination with the appropriate wavelength of light or removal of the appropriate wavelength of light. The engineered HEK293 cells further comprise the adenoviral helper genes encoding adenovirus E4 protein, adenovirus E2A protein, and adenovirus VA RNA.

[0224] Optionally, all four AAV Rep proteins are under the transcriptional control of the same light controllable transcriptional regulator. First, a transposon donor plasmid is generated via Gibson assembly of DNA molecules. The transposon donor plasmid encodes a light-controllable transcriptional regulator and a polycistronic gene that encodes a polypeptide comprising an AAV8 Rep78 protein, an AAV8 Rep68 protein, an AAV8 Rep 52 protein and an AAV8 Rep40 protein separated by self-cleaving T2A peptides, wherein expression of the polycistronic gene is operably linked by a light inducible promoter. Optionally, the transposon donor plasmid encodes Rep78 / 68 and Rep52 / 40 separated by a self-cleaving T2A peptide, wherein expression of the polycistronic gene is operably linked by a light inducible promoter.

[0225] Optionally, all three AAV Capsid proteins are under the transcriptional control of the same light controllable transcriptional regulator. First, a transposon donor plasmid is generated via Gibson assembly of DNA molecules. The transposon donor plasmid encodes a light-controllable transcriptional regulator and the AAV8 Cap gene that comprises the coding sequences of AAV8 VP1 protein, AAV8 VP2 protein, and AAV8 VP3 protein, wherein expression of the AAV8 Cap gene is operably linked by a light inducible promoter.

[0226] Optionally, the adenovirus helper genes encoding adenovirus E4 protein, adenovirus E2A protein, and adenovirus VA RNA are also under the control of a light-inducible promoter.WSGR Docket No. 60885-743.601

[0227] For engineering of the HEK293 cells, the cells are transfected with all required transposon donor plasmids and a transposase (e.g. encoded on a plasmid or mRNA) to allow stable integration. The engineered cell reversibly expresses all four AAV8 Rep proteins and / or all three AAV8 Capsid, and / or a subset or all adenovirus helper genes upon illumination with the appropriate wavelengths of light or removal of the appropriate wavelengths of light.Generating rAA V particles

[0228] The engineered cells are then transfected with plasmid AAV-CMV-EGFP, which comprises a nucleotide sequence encoding an rAAV genome. The transfected cells are then stimulated with the appropriate wavelengths of light to trigger expression of the AAV Rep and Cap proteins (and optionally the adenovirus helper genes) resulting in the formation of rAAV8 particles.Collecting rAAV particles

[0229] The cell culture containing rAAV8 particles is collected 4 days post-onset of illumination with the appropriate wavelength of light. Both the cells (pelleted by centrifugation) and the cell supernatant are harvested.Purifying and concentrating rAAV particles

[0230] The rAAV particles are precipitated from the supernatant by adding polyethylene glycol (PEG), gently mixing the solution, and incubating the solution at 4°C. The solution is then centrifuged to generate a PEG pellet containing the rAAV particles. The cell pellet is resuspended in a high salt buffer comprising a salt-activated nuclease to lyse any intact cells and digest any DNA not encapsidated in an rAAV capsid. The PEG pellet is also resuspended in a high salt buffer comprising a salt-activated nuclease to lyse any intact cells and digest any DNA not encapsidated in an rAAV capsid. The resuspended PEG pellets and cell pellets are pooled. The pooled pellets are purified and concentrated using iodixanol gradient ultracentrifugation. The fraction containing the purified rAAV particles is collected, and any residual iodixanol is removed via buffer exchange using an Amicon filtration device.AAV titration via ddPCR

[0231] The purified and concentrated rAAV particles are titrated via ddPCR using primers complimentary to the AAV internal terminal repeat sequences.Determining the percentage of empty AA V particles

[0232] The percentage of empty AAV particles is determined using Transmission electron microscopy (TEM). The empty capsid ratio of the purified and concentrated rAAV particles is less than 95%.Assessing rAA V transduction efficiency in vitroWSGR Docket No. 60885-743.601

[0233] HepG2 (human hepatocellular carcinoma cells), Huh7 (human hepatocellular carcinoma cells), C2C12 (mouse myoblast cells), and HEK293 cells are seeded at a density of 5,000 cells cm -2 in a 96-well plate. After 24 h, viral vectors at different MOI (1, 10, 100, 1,000, 10,000, 100,000, 1,000,000) are added and after another 48 h the transduction efficiency of the rAAV8 particles for the different cell lines is assessed by measuring the percentage of cells that express eGFP via flow cytometry.Example 3 - Producing rAAV particles using producer cells

[0234] rAAV8 particles are created by first generating a producer cell comprising adenoviral helper genes, an rAAV genome, and at least two gene expression cassettes, the first encoding AAV8 Rep proteins and the second encoding AAV8 Capsid proteins, each under the transcriptional control of a corresponding light inducible promoter. For production of viral vectors, expression of the AAV Rep and Cap proteins is induced upon illumination with the appropriate wavelengths of light or removal of the appropriate wavelengths of light.

[0235] The produced rAAV8 particles express eGFP under the transcriptional control of a ubiquitous CMV promoter. The rAAV8 particles generated by the methods described herein are used to further assess the transduction efficiency of rAAV8 in vitro.Generating a producer cell that produces rAA V8 particles when illuminated with appropriate wavelengths of light.

[0236] First, nine transposon donor plasmids are generated via Gibson assembly of DNA molecules. The first transposon donor plasmid encodes a first light-controllable transcriptional regulator and an AAV8 Rep78 protein operably linked by a first light inducible promoter. The second transposon donor plasmid encodes a second light-controllable transcriptional regulator and an AAV8 Rep68 protein operably linked by a second light inducible promoter. The third transposon donor plasmid encodes a third light-controllable transcriptional regulator and an AAV8 Rep52 protein operably linked by a third light inducible promoter. The fourth transposon donor plasmid encodes a fourth light-controllable transcriptional regulator and an AAV8 Rep40 protein operably linked by a fourth light inducible promoter. The fifth transposon donor plasmid encodes a fifth light-controllable transcriptional regulator and an AAV8 VP1 protein operably linked by a fifth light inducible promoter. The sixth transposon donor plasmid encodes a sixth light-controllable transcriptional regulator and an AAV8 VP2 protein operably linked by a sixth light inducible promoter. The seventh transposon donor plasmid encodes a seventh light-controllable transcriptional regulator and an AAV8 VP3 protein operably linked by a seventh light inducible promoter. The eighth transposon donor plasmid encodes the adenoviral helper genesWSGR Docket No. 60885-743.601encoding adenovirus E4 protein, adenovirus E2A protein, and adenovirus VA RNA. The ninth transposon donor plasmid encodes an rAAV genome.

[0237] For engineering of the HEK293 cells, the cells are transfected with each of the nine transposon donor plasmids and a transposase (e.g. encoded on a plasmid or mRNA) to allow stable integration.

[0238] The engineered HEK293 cells comprise 7 gene cassettes, each capable of expressing AAV8 Rep78, AAV8 Rep68, AAV8 Rep52, AAV8 Rep40, AAV8 VP1, AAV8 VP2 or AAV8 VP3 protein by activating a corresponding light controllable transcriptional regulator upon illumination with the appropriate wavelengths of light. The engineered cell reversibly expresses each of the AAV8 Rep and / or AAV8 Cap proteins upon illumination with the appropriate wavelength of light or removal of the appropriate wavelength of light. The engineered HEK293 cells further comprise the rAAV genome and the adenoviral helper genes encoding adenovirus E4 protein, adenovirus E2A protein, and adenovirus VA RNA.

[0239] Optionally, all four AAV Rep proteins are under the transcriptional control of the same light controllable transcriptional regulator. First, a transposon donor plasmid is generated via Gibson assembly of DNA molecules. The transposon donor plasmid encodes a light-controllable transcriptional regulator and a polycistronic gene that encodes a polypeptide comprising an AAV8 Rep78 protein, an AAV8 Rep68 protein, an AAV8 Rep 52 protein and an AAV8 Rep40 protein separated by self-cleaving T2A peptides, wherein expression of the polycistronic gene is operably linked by a light inducible promoter. Optionally, the transposon donor plasmid encodes Rep78 / 68 and Rep52 / 40 separated by a self-cleaving T2A peptide, wherein expression of the polycistronic gene is operably linked by a light inducible promoter.

[0240] Optionally, all three AAV Capsid proteins are under the transcriptional control of the same light controllable transcriptional regulator. First, a transposon donor plasmid is generated via Gibson assembly of DNA molecules. The transposon donor plasmid encodes a light-controllable transcriptional regulator and the AAV8 Cap gene that comprises the coding sequences of AAV8 VP1 protein, AAV8 VP2 protein, and AAV8 VP3 protein, wherein expression of the AAV8 Cap gene is operably linked by a light inducible promoter.

[0241] Optionally, the adenovirus helper genes encoding adenovirus E4 protein, adenovirus E2A protein, and adenovirus VA RNA are also under the control of a light-inducible promoter.

[0242] For engineering of the HEK293 cells, the cells are transfected with all required transposon donor plasmids and a transposase (e.g., encoded on a plasmid or mRNA) to allow stable integration. The engineered cell reversibly expresses all four AAV8 Rep proteins and / or all three AAV8 Capsid, and / or a subset or all adenovirus helper genes upon illumination with the appropriate wavelengths of light or removal of the appropriate wavelengths of light.WSGR Docket No. 60885-743.601Generating rAA V particles

[0243] The transfected cells are then stimulated with the appropriate wavelengths of light to trigger expression of the AAV Rep and Cap proteins (and optionally the adenovirus helper genes) resulting in the formation of rAAV8 particles.Collecting rAAV particles

[0244] The cell culture containing rAAV8 particles is collected 4 days post-onset of illumination with the appropriate wavelength of light. Both the cells (pelleted by centrifugation) and the cell supernatant are harvested.Purifying and concentrating rAAV particles

[0245] The rAAV particles are precipitated from the supernatant by adding polyethylene glycol (PEG), gently mixing the solution, and incubating the solution at 4°C. The solution is then centrifuged to generate a PEG pellet containing the rAAV particles. The cell pellet is resuspended in a high salt buffer comprising a salt-activated nuclease to lyse any intact cells and digest any DNA not encapsidated in an rAAV capsid. The PEG pellet is also resuspended in a high salt buffer comprising a salt-activated nuclease to lyse any intact cells and digest any DNA not encapsidated in an rAAV capsid. The resuspended PEG pellets and cell pellets are pooled. The pooled pellets are purified and concentrated using iodixanol gradient ultracentrifugation. The fraction containing the purified rAAV particles is collected, and any residual iodixanol is removed via buffer exchange using an Amicon filtration device.AAV titration via ddPCR

[0246] The purified and concentrated rAAV particles are titrated via ddPCR using primers complimentary to the AAV internal terminal repeat sequences.Determining the percentage of empty AA V particles

[0247] The percentage of empty AAV particles is determined using Transmission electron microscopy (TEM). The empty capsid ratio of the purified and concentrated rAAV particles is less than 95%.Assessing rAA V transduction efficiency in vitro

[0248] HepG2 (human hepatocellular carcinoma cells), Huh7 (human hepatocellular carcinoma cells), C2C12 (mouse myoblast cells), and HEK293 cells are seeded at a density of 5,000 cells cm -2 in a 96-well plate. After 24 h, viral vectors at different MOI (1, 10, 100, 1,000, 10,000, 100,000, 1,000,000) are added and after another 48 h the transduction efficiency of the rAAV8 particles for the different cell lines is assessed by measuring the percentage of cells that express eGFP via flow cytometry.Example 4 - Example protocol for producing and characterizing AAV particlesWSGR Docket No. 60885-743.601MethodsCell Culture

[0249] Suspension HEK293F cells (Viral Production Cells 2.0 (VPC2.0), Thermo Fisher Scientific) were maintained in Viral Production Medium (Thermo Fisher Scientific) supplemented with GlutaMAX (Gibco) at a final concentration of 4 mM. Cells were maintained at densities between 0.2e6 and 10e6 cells / mL via passaging. Suspension cells were shaken at 900 rpm with 3 mm throw in 96-deepwell plate culture conditions, 300 rpm with 19 mm throw for 24-deepwell plate culture conditions, and 130 rpm with 25 mm throw for flasks. All cell handling was performed under green or red safe light to prevent activation of the blue lightinducible gene expression system.Molecular Cloning

[0250] Constructs designed in this study were generated by DNA synthesis and cloned into a pUC19-derived plasmid via Gibson assembly. All constructs were sequence-verified using whole-plasmid sequencing.Transduction Assay

[0251] Approximately 1 mL of the cell suspension at the end of the AAV production run was lysed using AAV-MAX lysis buffer (Thermo Fisher Scientific) with 250U Pierce universal nuclease and 2 mM MgCh shaking at 37°C for Ih. Next, nuclease degradation was quenched by addition of 500 mM NaCl and 5 mM EDTA. Afterwards, cell debris was spun down at 1000 g for 5 min and the supernatant was added at a 100-fold final dilution to 5e3 VPC2 cells seeded in 100 pL viral production medium in a 96-well plate. After incubation for 2-3 days, transduced cells were quantified by assessing GFP fluorescence by flow cytometry.Genome (vg) Titer

[0252] To determine the viral genome (vg) titer, viral supernatant collected for transduction assay was diluted lOx into lx proteinase K buffer, and 50 U proteinase K at a final volume of 100 pl. The proteinase K treated viral vectors were incubated at 65°C for Ih and then proteinase K was inactivated at 95°C for lOmin. Afterwards, viral DNA was serially diluted and genomic titer was determined by ddPCR using the following primers (see Table 7) targeting the CMV promoter in the GOI plasmid.Table 7. Example primers / probesWSGR Docket No. 60885-743.601Western Blot

[0253] Approximately 0.8 mL of the cell suspension at the end of the AAV production run was harvested by centrifugation at 1000 g for 5min and the cells were lysed in 100 pL RIPA buffer spiked with 250U pierce universal nuclease and lx HALT protease inhibitor per sample. Samples were further diluted in lx Bolt Reducing agent and lx LDS sample buffer. Cell lysates were heat inactivated at 95°C and were separated on 4-12% Bolt NuPAGE gels using MOPS buffer. Gels were transferred using the Bio-Rad TurboBlot transfer system to low fluorescence 0.45 pm PVDF membranes. Blots were then blocked with Bio-Rad EveryBlot blocking buffer for 5 min and antibodies were diluted in blocking buffer. Primary antibodies were incubated for 1 h at room temperature and secondary antibodies were incubated for 30 min at room temperature. Blots were washed twice with TBS with 0.1% Tween-20 (TBST) for 2 min in between stains. HRP conjpgated antibodies were visualized using theSuperSignal West Atto Ultimate Sensitivity Substrate (Thermo Fisher Scientific) and imaged using the Bio-Rad ChemiDoc MP system. See Table 8 for example antibodies.Table 8. Non-Limiting Exemplary AntibodiesExample 5 - Generation of suspension HEK cell lines stably expressing the optogenetic gene expression system (OptoChassis)

[0254] Details provided throughout the present disclosure (e.g., any one of preceding and subsequent Examples) may be utilized herein.WSGR Docket No. 60885-743.601

[0255] To stably integrate an expression cassette for the eGAV#12 light-inducible gene expression system into suspension HEK293F cells (Viral Production Cells 2.0 (VPC2.0), Thermo Fisher Scientific), VPC2.0 cells were transfected with plasmid P 1285 or plasmid P_2074 and transposase mRNA using the AAV-MAX transfection kit (Thermo Fisher Scientific). See FIG. 9 for additional details for plasmid P 1285 and plasmid P 2074. FIG. 9 shows design of the plasmids containing the expression cassettes for the optogenetic gene expression system for the stable integration. The two plasmids differ in the identity of the transposon ITRs. The depicted cassettes were cloned into a pUC19-derived bacterial plasmid backbone. ITR, inverted terminal repeat. P Constl, constitutive promoter. p65AD, p65 transcriptional activation domain ofNF-KB. GAL4DBD, Gal4 DNA binding domain. VVD, Vivid photoreceptor. pA, polyadenylation signal.

[0256] To this aim, 2 mL of VPC2.0 cells were seeded at a concentration of 1.5e6 cells / mL the day before transfection. For transfection, 2.4 pg of plasmid was mixed with 0.6 pg of mRNA in 200 pL Viral-Plex complexation buffer. Next, 12 pL of AAV-MAX transfection reagent was mixed with 6 pL of AAV-MAX transfection booster and added to the complexation buffer. Following incubation for 25 min at room temperature, the transfection mix was added to 2 mL of the cells previously supplemented with 20 pL of the AAV-MAX enhancer. After 8 days, when the non-stably integrated plasmids were diluted out by cell division, the cells were transfected with a plasmid encoding a destabilized version of the fluorescent protein mScarlet under control of a light-inducible promoter and a plasmid encoding the fluorescent protein GFP under a constitutive promoter using the FectoVIR-AAV (Sartorius) transfection reagent. To this aim, 2 mL of cells were seeded at a concentration of 1.5e6 cells / mL the day before transfection. For transfection, 0.75 pg of the light-inducible mScarlet reporter plasmid, 0.5 pg of the constitutive GFP plasmid, and 0.75 pg of an empty pUC19 filler plasmid were mixed with 2 pL FectoVIR-AAV transfection reagent in 200 pl complexation buffer. After incubation for 25 min at room temperature, the transfection mix was added to 2 mL of the cells. 48 h after transfection, the cells were illuminated for 24 h with 470 nm blue light at an intensity of 2 pW / mm2Afterwards, both GFP- and mScarlet-positive cells were sorted on a Wolf G2 cell sorter.Following expansion of the sorted cells for 10 days, single cells were sorted using a Wolf G2 cell sorter into 96-well plates and expanded. Both the pools and the clones generated were screened for stable integration of the optogenetic gene expression system by transfecting with a plasmid encoding a destabilized version of the fluorescent protein mScarlet under a lightinducible promoter and a plasmid encoding the fluorescent protein GFP under a constitutive promoter using the FectoVIR-AAV (Sartorius) transfection reagent.WSGR Docket No. 60885-743.601

[0257] It was observed that in this screening assay -60% of the cells in the OptoChassis pool were responsive to light, whereas in the OptoChassis clones up to -85% of the cells were light-responsive (FIGs. 10A and 10B). FIGs. 10A and 10B show functional testing of the cell lines with stable integration of the optogenetic gene expression system (OptoChassis). The OptoChassis pool (transposon-mediated integration of plasmid P 2074) and three selected OptoChassis clones (D2, E9, and H4. Transposon-mediated integration of plasmid P 1285) were transfected with reporter plasmid driving expression of the fluorescent protein mScarlet under a light-inducible promoter and a plasmid constitutively expressing the fluorescent protein GFP. After illumination for 24 h with blue light, fluorescence was analyzed by flow cytometry.Scatter plots in FIG. 10A show mScarlet and GFP fluorescence of the cells after illumination and in the dark. FIG. 10B shows quantification of the percentage of light-responsive cells. Cells were gated on GFP -positive cells and the percentage of responsive cells in the light was quantified based on the mScarlet signal.Example 6 - Adenoviral helper proteins expression can be controlled by light in transient transfection and allows the production of functional AAV vectors

[0258] Details provided throughout the present disclosure (e.g., any one of preceding and subsequent Examples) may be utilized herein.

[0259] Four constructs were designed to express adenoviral helper proteins in response to light. All constructs share the following vector elements: flanking transposon ITR sites, and an mNeonGreen expression cassette comprising a constitutive promoter, mNeonGreen coding sequence, and a polyadenylation sequence. Briefly, these constructs are denoted as P 2405 (containing an adenoviral helper expression cassette comprising an inducible promoter comprising UAS element(s) and a minimal promoter followed by early region 2A (E2A) coding sequence, a T2A self-cleaving linker, early region 4 (E4) open reading frames 6 and 6 / 7 coding sequences, internal ribosome entry site 2 (IRES2), late transcription unit 4 (L4) coding sequences, and a polyadenylation sequence), P 2414 (containing an adenoviral helper expression cassette comprising an inducible promoter comprising UAS element(s) and a minimal promoter followed by early region 2A (E2A) coding sequence, a T2A self-cleaving linker, late transcription unit 4 (L4) coding sequence, and a polyadenylation sequence), P 2415 (containing an adenoviral helper expression cassette comprising an inducible promoter comprising UAS element(s) and a minimal promoter followed by early region 2A (E2A) coding sequence, a T2A self-cleaving linker, late transcription unit 4 (L4) coding sequences, a T2A self-cleaving linker, early region 4 (E4) open reading frames 6 and 6 / 7 coding sequences, and a polyadenylation sequence), and P 2420 (containing an adenoviral helper expression cassetteWSGR Docket No. 60885-743.601comprising an inducible promoter comprising UAS element(s) and a minimal promoter followed by early region 2A (E2A) coding sequence, a T2A self-cleaving linker, late transcription unit 4 (L4) coding sequences, internal ribosome entry site 2 (IRES2), early region 4(E4) open reading frame 6 and 6 / 7 coding sequence, and a polyadenylation sequence) (see FIG. 11 for additional details on abovementioned plasmids). FIG. 11 shows example design of the plasmids containing the light-inducible expression cassettes for the adenoviral helper proteins. The depicted cassettes were cloned into a pUC19-derived bacterial plasmid backbone. ITR, inverted terminal repeat. pA, polyadenylation sequence. P Induc, inducible promoter comprising UAS binding site and a minimal promoter. E2A, early region 2A coding sequence. T2A, self-cleaving linker. E4, early region 4 open reading frame 6 and spliced E4orf6 / 7 coding sequences. L4, late transcription unit 4 with 22k and 33k spliced coding sequences. IRES2, internal ribosome entry site 2. P_Const2, constitutive promoter. mNeonGreen, green fluorescent protein.

[0260] These plasmids were co-transfected into suspension HEK293F cells (Viral Production Cells 2.0 (VPC2.0), Thermo Fisher Scientific) stably expressing the optogenetic gene expression system (OptoChassis pool) with RepCap (P 2408, Genscript, cat. no. RP-B00003) and GOI (P 2412) plasmids. For the transfection, a molar ratio of 2:2: 1RepCap :GOI:Helper in a total plasmid mass of 4 pg was mixed in 200 pL Viral-Plex complexation buffer (Sartorius). Next, 4 pl of FectoVir transfection reagent (Sartorius) was added to the complexation buffer. Following incubation for 25 min at room temperature, 200 pL of the transfection mix was added to 2 mL of OptoChassis cells seeded at 2e6 cells / mL. As control, HEK293F cells (Viral Production Cells 2.0 (VPC2.0), Thermo Fisher Scientific) were transfected with a conventional Helper plasmid (P 2409, Genscript, cat. no. RP-B00001) and with RepCap (P 2408) and GOI (P 2412) plasmids at the same molar ratio. See FIG. 21 for additional details for plasmids P 2409, P 2408, and P 2412. FIG. 21 shows design of the plasmids used for production of GFP-encoding AAV vectors together with the light-inducible Rep and Helper constructs. Plasmid 2409 (pHelper) comprises adenovirus type 2 (Ad2) helper genes. Plasmid 2408 (RepCap) provides AAV2 rep and cap functions. Plasmid 2412 (GOI) encodes a self-complementary AAV vector genome (ITR-flanked EGFP payload). Plasmid 2406 (CapGOI) contains the same ITR-flanked payload cassette and additionally comprises a Cap2 expression cassette under control of the p40 promoter. ITR, inverted terminal repeat. pA, polyadenylation sequence. E2A, early region 2A. E4, early region 4. EGFP-HiBit, enhanced green fluorescent protein with a HiBit nanoluciferase tag. P CMV, constitutive cytomegalovirus promoter. Atrs, terminal resolution site deletion. Rep2_Cterm, C-terminal region of Rep2 gene comprising the p40 promoter. p5, pl 9, p40, adeno-associated viral promoters. Cells wereWSGR Docket No. 60885-743.601maintained in the dark or illuminated with 470 nm blue light at an intensity of 2.1 pW / mm2After 3 days, viral vectors were harvested for transduction assay, vg titer assay, and Western Blotting against E2A, Rep, and Cap as described above.

[0261] It was observed that AAV vectors produced using the light-inducible adenoviral helper constructs P 2405, P 2415, and P 2420 displayed an up to a 350-fold improvement in transduction efficiency and vg titer in the light compared to the dark (see FIG. 12A). Construct P 2414 did not result in production of functional AAV vectors in the dark and light. AAV vectors produced with the light-inducible adenoviral helper construct P 2405 exceeded transduction efficiency and vg titer of AAV vectors produced by triple transfection with a conventional adenoviral helper plasmid illuminated at the same intensity. Western Blot analysis demonstrated that all 4 constructs show E2A, Rep, and Cap expression in the light at significantly higher levels than observed in the dark (see FIG. 12B). FIGs. 12A and 12B show that adenoviral helper protein expression can be controlled by light in transient transfection and allows the production of functional AAV vectors. AAV vectors encoding GFP were produced by triple transfection of one of the light-inducible adenoviral helper plasmids 2405, 2414, 2415, or 2420 and the RepCap and GOI plasmid into suspension HEK293F cells stably expressing the optogenetic gene expression system (OptoChassis pool). As a positive control (+CTRL), a conventional adenoviral helper plasmid was used. Cells were incubated in the dark or illuminated with blue light (2.1 pW / mm2) for 3 days. In FIG. 12A, potency of the produced AAV vectors was determined in a transduction assay (upper panel) and the vg titer was determined by ddPCR (lower panel). FIG. 12B shows Western Blot analysis of the production cells against E2A, Rep, and Cap (VP1 / VP2 / VP3) proteins.Example 7 - Temporal control of adenoviral helper protein expression improves titer compared to constitutive expression at different levels

[0262] Details provided throughout the present disclosure (e.g., any one of preceding and subsequent Examples) may be utilized herein.

[0263] The light-inducible adenoviral helper plasmid P 2405 was co-transfected into suspension HEK293F cells (Viral Production Cells 2.0 (VPC2.0), Thermo Fisher Scientific) stably expressing the optogenetic gene expression system (OptoChassis clone E9) with RepCap (P 2408) and GOI (P 2412) plasmids. For the transfection, a molar ratio of 2:2: 1RepCap :GOI:Helper in a total plasmid mass of 80 pg was mixed in 4.2 mL Viral-Plex complexation buffer. Next, 80 pL of FectoVir transfection reagent was added to the complexation buffer. Following incubation for 25 min at room temperature, 4 mL of the transfection mix was added to 40 mL of OptoChassis clone E9 cells seeded at 2e6 cells / mL in aWSGR Docket No. 60885-743.601flask. After incubation for 6 hours in the dark, cells were seeded from the flask into a 24-deepwell plate and exposed to different illumination regimes as described in FIGs. 12A and 12B and Example 6. After 3 days, viral vectors were harvested for transduction assay.

[0264] It was observed novel and surprising effects of the different temporal adenoviral helper expression patterns on the produced AAV vector titer (FIG. 13). FIG. 13 shows that temporal control of adenoviral helper protein expression improves titer compared to constitutive expression at different levels. AAV vectors encoding GFP were produced by triple transfection of the light-inducible adenoviral helper plasmid 2405 and the RepCap and GOI plasmids into suspension HEK293F cells stably expressing the optogenetic gene expression system (OptoChassis clone E9). 6 h after transfection the cells were exposed to different illumination regimes for a total duration of 72 h. Afterwards, potency of the produced AAV vectors was determined in a transduction assay.

[0265] First, it was observed that Helper expression is critical in the first 24 h after transfection as all light patterns in which this time period was dark only produced comparably low amounts of functional AAV vectors. Next, it was observed that switching off Helper expression after 12 h, 24 h, and 48 h was able to improve titer compared to constant Helper expression. It was observed that the light pattern in which Helper was induced for 48 h with blue light (2.1 pW / mm2) and then switched off for the final 24 h showed 61% higher transduction titer than constant blue illumination for 72 h at 1.4 pW / mm2intensity (equivalent overall light dose). Importantly, removal of the inducer is impractical using conventional chemically inducible gene expression systems, demonstrating a unique advantage of optogenetic induction of Helper protein expression.Example 8 - Generation of stable light-inducible Rep40 / 68 cell lines which allow production of functional AAV vectors in response to light

[0266] Details provided throughout the present disclosure (e.g., any one of preceding and subsequent Examples) may be utilized herein.

[0267] A plasmid (P 2402) containing a light-inducible expression cassette for the Rep40 and Rep68 proteins was designed. See FIG. 14 for additional details for plasmid P 2402.FIG. 14 shows design of the plasmids containing the light-inducible expression cassettes for the Rep proteins used for stable integration. The depicted cassettes were cloned into a pUC19-derived bacterial plasmid backbone. ITR, inverted terminal repeat. pA, polyadenylation sequence. P Induc, inducible promoter comprising UAS binding site and a minimal promoter. T2A, self-cleaving linker. P_Const2, constitutive promoter. mScarlet, red fluorescent protein.WSGR Docket No. 60885-743.601

[0268] Briefly, this construct comprises flanking transposon ITR sites, a light-inducible Rep expression cassette comprising an inducible promoter comprising UAS element(s) and a minimal promoter followed by the coding sequence of Rep40, a T2A self-cleaving linker, the coding sequence of Rep68, and a polyadenylation sequence, and an mScarlet expression cassette comprising a constitutive promoter, mScarlet-I3 coding sequence, and a polyadenylation sequence. The last 231 nucleotides of Rep40 and Rep68 were codon-optimized to ablate the p40 promoter activity. A M225G mutation was introduced into Rep68 to ablate the native start codon of Rep40 within Rep68.

[0269] To stably integrate P 2402 into suspension HEK293F cells (Viral Production Cells 2.0 (VPC2.0), Thermo Fisher Scientific) stably expressing the optogenetic gene expression system (OptoChassis), OptoChassis pools with integration of the optogenetic gene expression system were transfected with plasmid P_2402 and transposase mRNA using the AAV-MAX transfection kit (Thermo Fisher Scientific). To this aim, 2 mL of OptoChassis pool cells were seeded in 500 pL at a concentration of 1.5e6 cells / mL the day before transfection. For transfection, 2.4 pg of plasmid was mixed with 0.6 pg of mRNA in 200 pL Viral-Plex complexation buffer. Next, 12 pL of AAV-MAX transfection reagent was mixed with 6 pl of AAV-MAX transfection booster and added to the complexation buffer. Following incubation for 25 min at room temperature, 50 pL of the transfection mix was added to 500 pL of the cells previously supplemented with 5 pl of the AAV-MAX enhancer.

[0270] After 11 days, when the non-stably integrated plasmids were diluted out by cell division, mScarlet-positive cells were single-cell sorted on a Wolf G2 cell sorter into 96-well plates and expanded. After expansion in static 96-well plates for 14 days, clones were transferred to static 24-well plates. After expansion in static 24-well plates for 7 days, clones were assayed for mScarlet expression via flow cytometry.

[0271] Next, 36 mScarlet-positive clones (24 from the OptoChassis pool generated from P 1285 integration and 12 from the OptoChassis pool generated from P 2074 integration) were screened for light-inducible Rep expression. To this aim, ~5e4 cells of each clone were seeded into two wells of a static 96-well plate and either incubated in the dark or under illumination with 470 nm blue light at an intensity of 2.5 pW / mm2for 72 h. Afterwards, cells were harvested, and Rep expression was assessed by Western blotting. It was observed that the majority of the clones expressed Rep40 and Rep68 at significantly higher expression levels in the light as compared to the dark condition (FIG. 15). FIG. 15 shows screening of clones for light-inducible expression of Rep40 / 68 by Western Blotting. Suspension HEK293F cells were engineered to express Rep40 / 68 in response to blue light illumination and were clonally expanded. 36 clones (Clone T-l - clone T-24 from the OptoChassis pool generated from P 1285 and clone P-1 -WSGR Docket No. 60885-743.601clone P-12 from the OptoChassis pool generated from P 2074) were assessed for expression of Rep proteins in the dark and in response to illumination with 470 nm blue light at an intensity of 2.5 pW / mm2for 72 h. Based on the results of the Western blot, the clones T-2, T-12, and P-8 with light-inducible Rep40 / 68 expression were selected for further testing and transferred to shaking culture.

[0272] FIGs. 16A-C show characterization of cell viability and proliferation in the dark and light for the clones T-2, T-12, and P-8 with light-inducible Rep40 / 68 expression. As control, non-engineered VPC2.0 cells were used. FIG. 16A shows assessment of the doubling time in the dark for 2 passages. FIG. 16B shows analysis of the cell viability after 4 days in the dark or under constant 470 nm blue light illumination at an intensity of 2.5 pW / mm2by DAPI staining and flow cytometry analysis. FIG. 16C shows analysis of the doubling time between day 1 and day 3 after illumination with constant 470 nm blue light illumination at an intensity of 0.5 pW / mm2or incubation in the dark.

[0273] First, the proliferation rate of these clones in the absence of light induction was assessed. Doubling times over 2 passages were calculated and compared against the nonengineered VPC2.0 cells. It was observed that clone T-12 and P-8 showed a comparable doubling time to the VPC2.0 cells whereas the clone T-2 showed a slightly increased doubling time of 40 h (FIG. 16A). This demonstrates that the Rep expression in clones T-12 and P-8 in the uninduced dark state is low enough to not negatively affect cell proliferation.

[0274] Next, the impact of Rep expression on cell viability and proliferation was analyzed. To assess the impact on cell viability, le4 cells of each clone and of VPC2.0 cells were seeded per well of a static 96-well plate. After incubation for 4 days in the dark or under constant 470 nm blue light illumination at an intensity of 2.5 pW / mm2, cell viability was assessed by DAPI staining and flow cytometry. It was observed that the light-induced expression of Rep40 / 68 resulted in high cell death (i.e. DAPI-positive cells) for the clones T-2, T-12, and P-8 whereas the viability of the clones in the dark was comparable to the viability of non-enginnered VPC2.0 cells in dark and light. To assess the impact on cell proliferation, le4 cells of each clone and of VPC2.0 cells were seeded per well of a static 96-well plate and incubated in the dark or exposed to constant 470 nm blue light at an intensity of 0.5 pW / mm2for 3 days. Cells were counted at day 1 and day 3 via flow cytometry and the doubling times between the two timepoints were calculated. It was observed that the clones T-2, T-12, and P-8 showed a significant increase in doubling time in the light compared to the dark whereas the impact of the light on the non-engineered VPC2.0 cells was marginal. Clone T-12 showed the highest Rep expression-induced increase in doubling time with an average doubling time of -30 h in the dark and -450 h in the light. This data demonstrates that the light-induced RepWSGR Docket No. 60885-743.601expression levels in the clones T-2, T-12, and P-8 have a strong negative impact on cell viability and cell proliferation. See FIG. 16B (cell viability) and FIG. 16C (cell proliferation).

[0275] Finally, the clones T-2, T-12, and P-8 with light-inducible Rep40 / 68 expression were assayed for their ability to produce functional AAV when transfected with CapGOI (P 2406) and light-inducible Helper (P 2405) plasmids and induced with light. See FIG. 21 for additional details for plasmid P 2406. To this end, 2 mL of the cells were seeded at 2e6 cells / mL in 24-deepwell plates on the day of transfection. For transfection, 4 pg of Cap-GOI and Helper plasmids at a molar ratio of 2: 1 were mixed in 200 pL Viral-Plex complexation buffer. Next, 4 pL of FectoVir transfection reagent was added to the complexation buffer. Following incubation for 25 min at room temperature, 200 pL of the transfection mix was added to 2 mL of the cells. Cells were either maintained in the dark or maintained in the dark for 6 h and subsequently illuminated with 470 nm blue light at an intensity of 2.1 pW / mm2After 3 days, viral vectors were harvested for transduction assay and Western Blotting against Rep as described above.

[0276] FIGs. 17A and 17B show that stable clones with light-inducible Rep40 / 68 expression allow the production of functional AAV vectors upon illumination. AAV vectors encoding GFP were produced by transfection of the light-inducible adenoviral helper plasmid 2405 and the Cap-GOI plasmid into the stable clones T-2, T-12, and P-8 with light-inducible Rep40 / 68 expression. Cells were incubated in the dark or illuminated with blue light (2.1 pW / mm2) for 3 days. FIG. 17A shows that potency of the produced AAV vectors was determined in a transduction assay. FIG. 17B shows the Western Blot analysis of the production cells against Rep proteins.

[0277] It was observed that AAV vectors produced from all 3 of the clones showed a higher transduction titer in the light than in the dark (FIG. 17A). Additionally, on Western blot (see FIG. 17B), all 3 clones showed markedly higher levels of Rep68 in the light as compared to the dark. Clone T-12 and Clone P-8 further showed higher levels of Rep40 in the light as compared to the dark. This data demonstrates that Rep expression is light-inducible and that the stable light-inducible Rep lines produce high enough levels of Rep40 / 68 in response to light to produce functional AAV vectors.Example 9 - Generation of stable light-inducible Rep52 / 78 cell lines which allow production of functional AAV vectors in response to light

[0278] Details provided throughout the present disclosure (e.g., any one of preceding and subsequent Examples) may be utilized herein.WSGR Docket No. 60885-743.601

[0279] Two plasmids were designed to express Rep52 and Rep78 proteins in response to light. All constructs share the following vector elements: flanking transposon ITR sites, and an mScarlet expression cassette comprising a constitutive promoter, mScarlet coding sequence, and a polyadenylation sequence. Briefly, these constructs are denoted as P 2427 (containing a Rep expression cassette comprising an inducible promoter comprising UAS element(s) and a minimal promoter followed by Rep52, a T2A self-cleaving linker, Rep78 containing an M225G mutation to ablate the native start codon for the internal small Rep protein, and a polyadenylation sequence) and P 2428 (containing a Rep expression cassette comprising an inducible promoter comprising UAS element(s) and a minimal promoter followed by Rep52, a T2A self-cleaving linker, Rep78 where the native start codon for the internal small Rep protein coding sequence remains, and a polyadenylation sequence) (See FIG. 14 for additional details for plasmid P 2427 and plasmid P 2428). The last 486 nucleotides of Rep52 and Rep78 were codon-optimized to ablate the p40 promoter activity.

[0280] To stably integrate P 2427 and P 2428 into suspension HEK293F cells (Viral Production Cells 2.0 (VPC2.0), Thermo Fisher Scientific) stably expressing the optogenetic gene expression system (OptoChassis clone E9), cells were transfected with with plasmid P_2427 or P_2428 and transposase mRNA using the AAV-MAX transfection kit (Thermo Fisher Scientific). To this aim, 500 pL of clone E9 cells were seeded at a concentration of 3e6 cells / mL on the day of transfection. For transfection, 2.4 pg of plasmid was mixed with 0.6 pg of mRNA in 200 pl Viral-Plex complexation buffer. Next, 12 pl of AAV-MAX transfection reagent was mixed with 6 pl of AAV-MAX transfection booster and added to the complexation buffer. Following incubation for 25 min at room temperature, 50 pL of the transfection mix was added to 500 pL of the cells previously supplemented with 5 pl of the AAV-MAX enhancer. After 11 days, when the non-stably integrated plasmids were diluted out by cell division, mScarlet positive cells were sorted on a Wolf G2 cell sorter and transferred to 96-deepwell plates. After expansion for 6 days, cells were transferred to 24-deepwell plates.

[0281] To assess whether the two generated stable light-inducible Rep52 / 78 pools allow production of functional AAV vectors in response to light, cells were transfected with CapGOI (P 2406) and inducible Helper (P 2405) plasmids at a 2:1 molar ratio. 4 pg total of DNA was mixed in 200 pL Viral-Plex complexation media. Next, 4 pL of FectoVir transfection Reagent was added to the complexation buffer. Following incubation for 25 min at room temperature, 200 pL of the transfection mix was added to 2 mL of the cells seeded at 2e6 cells / mL. Cells were maintained in the dark or illuminated with 470 nm blue light at an intensity of 2.1 pW / mm2After 3 days, viral vectors were harvested for transduction assay as described above.WSGR Docket No. 60885-743.601

[0282] FIG. 18 shows that stable pools with light-inducible Rep52 / 78 expression allow the production of functional AAV vectors upon illumination. AAV vectors encoding GFP were produced by transfection of the light-inducible adenoviral helper plasmid 2405 and the CapGOI plasmid into the stable pools with light-inducible Rep52 / 78 expression generated with plasmid P 2427 or P 2428. Cells were incubated in the dark or illuminated with blue light (2.1 pW / mm2) for 3 days. Potency of the produced AAV vectors was determined in a transduction assay.

[0283] It was observed that AAV vectors produced from both stable pools with inducible Rep52 / 78 expression showed a higher transduction titer in the light than in the dark (FIG. 18).The cell line without the M225G mutation (P 2428) generated showed higher transduction in light than the line with the mutation (P 2427) without an increase in transduction in the dark, suggesting that the small Rep isoform production within the Rep78 sequence may boost AAV titer in this configuration. This data demonstrates that Rep production is light-inducible and that the stable light-inducible Rep lines produce high enough levels of Rep52 / 78 in response to light to produce functional AAV vectors.Example 10 - Rep proteins expression can be controlled by light in transient transfection

[0284] Details provided throughout the present disclosure (e.g., any one of preceding and subsequent Examples) may be utilized herein.

[0285] Nine constructs were designed to express Rep proteins in response to light. All constructs share the following vector elements: flanking transposon ITR sites, a Rep expression cassette comprising an inducible promoter comprising UAS element(s) and a minimal promoter followed by the Rep cassette coding sequence, and a polyadenylation sequence, and a mScarlet or mNeonGreen expression cassette comprising a constitutive promoter, mScarlet or mNeonGreen coding sequence, and a polyadenylation sequence. Briefly, these constructs are denoted as P 2436 (comprising Rep40 with codon optimization 1), P 2437 (comprising Rep40 with codon optimization 2), P 2438 (comprising Rep52 with codon optimization 1), P 2439 (comprising Rep52 with codon optimization 2), P 2440 (comprising Rep68 with codon optimization 1), P 2441 (comprising Rep68 with codon optimization 2), P 2442 (comprising Rep78 with codon optimization 1), P 2443 (comprising Rep78 with codon optimization 2), P_2444 (comprising Rep52 with codon optimization 1, T2A self-cleaving linker, Rep78 with codon optimization 1). In all Rep52 and Rep78 coding sequences, the M225G mutation was introduced to ablate the native start codon for the internal small Rep protein. See FIG. 19 for additional details for the abovementioned plasmids. FIG. 19 shows design of the plasmids containing the light-inducible expression cassettes for the Rep proteins used for transientWSGR Docket No. 60885-743.601production. The depicted cassettes were cloned into a pUC19-derived bacterial plasmid backbone. ITR, inverted terminal repeat. pA, polyadenylation sequence. P Induc, inducible promoter comprising UAS binding site and a minimal promoter. T2A, self-cleaving linker. P_Const2, constitutive promoter. mScarlet, red fluorescent protein. mNeonGreen, green fluorescent protein.

[0286] These plasmids were co-transfected into suspension HEK293F cells (Viral Production Cells 2.0 (VPC2.0), Thermo Fisher Scientific) stably expressing the optogenetic gene expression system (OptoChassis pool) with conventional Helper (P 2409) and CapGOI (P_2406) plasmids.

[0287] For transfection, 4 pg total DNA in a molar ratio of 2: 1 : 1 : 1CapGOFRepl :Rep2:Helper for conditions with Rep proteins on 2 plasmids, and a molar ratio of 2:2: 1 CapGOI:Rep:Helper for conditions with both Rep proteins on one plasmid. Plasmids were mixed in 200 pL Viral -Plex complexation media. Next, 4 pL of FectoVir transfection Reagent was added to the complexation buffer. Following incubation for 25 min at room temperature, 200 pL of the transfection mix was added to 2 mL of the cells seeded at 2e6 cells / mL. Cells were maintained in the dark or illuminated with 470 nm blue light at an intensity of 2.1 pW / mm2After 3 days, viral vectors were harvested for transduction assay as described above.

[0288] Fig. 20 shows that Rep protein expression can be controlled by light in transient transfection and allows the production of functional AAV vectors. AAV vectors encoding GFP were produced by co-transfection of the depicted light-inducible Rep plasmids, a CapGOI plasmid, and a conventional Helper plasmid into suspension HEK293F cells stably expressing the optogenetic gene expression system (OptoChassis pool). Cells were incubated in the dark or illuminated with blue light (2.1 pW / mm2) for 3 days. Potency of the produced AAV vectors was determined in a transduction assay.

[0289] It was observed that a subset of experimental conditions were able to generate functional viral vectors in response to light induction. Induction of light-inducible AAV production was highly dependent on Rep protein codon optimization and Rep protein isoform. Codon optimization 1 was superior to codon optimization 2 (e.g., see FIG. 20 for comparison between (i) combination of P 2438 and P 2442 and (ii) combination of P 2439 and P 2443), and Rep52 / 78 was superior to Rep40 / 68 (e.g., see FIG. 20 for comparison between (i) combination of P 2438 and P 2442 and (ii) combination of P 2436 and P 2440). Multiple plasmid combinations showed a significant boost in titer of the produced AAV vectors in light compared to the dark (e.g., see FIG. 20 for (i) combination of P 2438 and P 2442, (ii) P 2444, and (iii) combination of P 2439 and P 2442). This data demonstrates that Rep protein expression can be controlled by light during AAV production via transient transfection.WSGR Docket No. 60885-743.601EMBODIMENTS

[0290] The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.Embodiment 1. A method of producing at least one viral protein, the method comprising:(a) providing or obtaining a cell comprising at least one exogenous nucleic acid comprising at least one nucleic acid sequence operably linked to a promoter and encoding at least one viral protein, and(b) controlling expression of the at least one viral protein using light.Embodiment 2. The method of Embodiment 1, wherein (b) comprises controlling the expression of the at least one viral protein via a temporary modulated exposure of the cell to the light.Embodiment 3. The method of Embodiment 2, wherein the temporary modulated exposure is provided by supplying the light to the cell and subsequently removing the light from the cell. Embodiment 4. The method of Embodiment 2 or Embodiment 3, wherein the temporary modulated exposure effects transient expression of the at least one viral protein in the cell.Embodiment s. The method of any one of Embodiment 2 to Embodiment 4, wherein a duration of the temporary modulated exposure is between about 0.5 hours and 60 hours.Embodiment 6. The method of Embodiment 5, wherein the duration of the temporary modulated exposure is between about 6 hours and 60 hours.Embodiment 7. The method of Embodiment 5, wherein the duration of the temporary modulated exposure is between about 10 hours and 60 hours.Embodiment 8. The method of Embodiment 5, wherein the duration of the temporary modulated exposure is between about 24 hours and 60 hours.Embodiment 9. The method of any one of Embodiment 2 to Embodiment 8, wherein the temporary modulated exposure comprises a continuous temporary modulated exposure.Embodiment 10. The method of any one of Embodiment 2 to Embodiment 9, further comprising culturing the cell for a culture period to produce a virus particle.Embodiment 11. The method of Embodiment 10, wherein the temporary modulated exposure is initiated during the initial 50% of the culture period.Embodiment 12. The method of Embodiment 10, wherein the temporary modulated exposure is initiated during the initial 30% of the culture period.Embodiment 13. The method of Embodiment 10, wherein the temporary modulated exposure is initiated within the initial 36 hours of the culture period.WSGR Docket No. 60885-743.601Embodiment 14. The method of Embodiment 10, wherein the temporary modulated exposure is initiated within the initial 24 hours of the culture period.Embodiment 15. The method of any one of Embodiment 10 to Embodiment 14, wherein a duration of the temporary modulated exposure is shorter than the culture period by at least about 10%, at least about 20%, or at least about 30%.Embodiment 16. The method of any one of Embodiment 10 to Embodiment 15, wherein the temporary modulated exposure of the cell to the light yields a production level of the virus particle that is greater than that produced via use of a comparable temporary modulated exposure to the light that is initiated at a later time point during the culture period than the temporary modulated exposure.Embodiment 17. The method of Embodiment 16, wherein the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at least about 10%, at least about 50%, at least about 100%, or at least about 200%.Embodiment 18. The method of Embodiment 16, wherein the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at least about 1-fold, at least about 2-fold, at least about 3-fold, or at least about 5 -fold.Embodiment 19. The method of any one of Embodiment 2 to Embodiment 18, wherein the temporary modulated exposure of the cell to the light yields a production level of the virus particle that is greater than that produced by via use of a longer exposure to the light.Embodiment 20. The method of Embodiment 19, wherein the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at least about 10%, at least about 50%, at least about 100%, or at least about 200%.Embodiment 21. The method of Embodiment 19, wherein the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at least about 0.5-fold, at least about 1-fold, or at least about 2-fold.Embodiment 22. The method of any one of Embodiment 1 to Embodiment 21, wherein the at least one nucleic acid sequence does not comprise a functional internal promoter associated with expression of the at least one viral protein in a native viral context.Embodiment 23. The method of Embodiment 22, wherein the at least one nucleic acid sequence does not comprise a gene encoding the functional internal promoter.Embodiment 24. The method of any one of Embodiment 1 to Embodiment 23, wherein the promoter of the at least one nucleic acid sequence is inducible by the light.WSGR Docket No. 60885-743.601Embodiment 25. The method of Embodiment 24, wherein the promoter is activated via a light controllable transcriptional regulator.Embodiment 26. The method of any one of Embodiment 1 to Embodiment 25, wherein the cell comprises an additional nucleic acid sequence encoding an additional promoter and an additional viral protein, wherein the additional promoter is not inducible by the light, and wherein the at least one viral protein and the additional viral protein are different.Embodiment 27. The method of Embodiment 26, wherein the cell comprises an additional exogenous nucleic acid comprising the additional nucleic acid sequence, wherein the at least one exogenous nucleic acid and the additional exogenous nucleic acid are different nucleic acid molecules.Embodiment 28. The method of Embodiment 26, wherein the cell comprises an additional exogenous nucleic acid comprising the additional nucleic acid sequence, wherein the at least one exogenous nucleic acid and the additional exogenous nucleic acid are within a single nucleic acid molecule.Embodiment 29. The method of any one of Embodiment 26 to Embodiment 28, wherein (i) the at least one viral protein comprises a helper protein and (ii) the additional viral protein comprises a replication protein, a capsid protein, or both.Embodiment 30. The method of Embodiment 29, wherein the helper protein comprises an adenovirus helper protein comprising one or more members selected from the group consisting of E2A, E4, and L4.Embodiment 31. The method of Embodiment 30, wherein the adenovirus helper protein comprises (i) E2A, (ii) E2A and E4, or (ii) E2A, E4, and L4.Embodiment 32. The method of any one of Embodiment 29 to Embodiment 31, wherein the additional promoter comprises a functional viral promoter.Embodiment 33. The method of Embodiment 32, wherein the functional viral promoter comprises p5, pl 9, or p40.Embodiment 34. The method of any one of Embodiment 26 to Embodiment 28, wherein (i) the at least one viral protein comprises a replication protein and (ii) the additional viral protein comprises a helper protein, a capsid protein, or both.Embodiment 35. The method of Embodiment 34, wherein the at least one viral protein comprises a large Rep protein and a small Rep protein.Embodiment 36. The method of Embodiment 35, wherein the at least one viral protein comprises Rep68 and Rep40.Embodiment 37. The method of Embodiment 35, wherein the at least one viral protein comprises Rep78 and Rep 52.WSGR Docket No. 60885-743.601Embodiment 38. The method of Embodiment 35, wherein the additional nucleic acid sequence comprises a nucleic acid molecule encoding the large Rep protein and a separate nucleic acid molecule encoding the small Rep protein.Embodiment 39. The method of any one of Embodiment 1 to Embodiment 38, wherein (i) at least a portion of the at least one nucleic acid sequence encoding the at least one viral protein is codon optimize, (ii) at least a portion of the additional nucleic acid sequence encoding the additional viral protein is codon optimized, or both.Embodiment 40. The method of any one of Embodiment 1 to Embodiment 39, wherein the cell further comprises at least one nucleic acid sequence encoding at least one light-controllable transcriptional regulator.Embodiment 41. The method of Embodiment 40, wherein the expression of the at least one viral protein is controllable by the at least one light-controllable transcriptional regulator.Embodiment 42. The method of any one of Embodiment 1 to Embodiment 41, wherein the promoter comprises an inducible promoter.Embodiment 43. The method of Embodiment 42, wherein the at least one light-controllable transcriptional regulator comprises a light-controllable transcriptional activator.Embodiment 44. The method of Embodiment 43, wherein the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain. Embodiment 45. The method of Embodiment 44, wherein illumination of the cell with light at a specific wavelength or wavelength range induces the light-controllable transcriptional activator to bind to and activate the inducible promoter or a binding domain thereof, thereby causing expression of the at least one viral protein.Embodiment 46. The method of Embodiment 44, wherein removing light or light at a specific wavelength or wavelength range from the cell induces the light-controllable transcriptional activator to bind to and activate the inducible promoter or a binding domain thereof, thereby causing expression of the at least one viral protein.Embodiment 47. The method of Embodiment 41, wherein the promoter is a constitutive promoter.Embodiment 48. The method of Embodiment 47, wherein the at least one light-controllable transcriptional regulator comprises a light-controllable transcriptional repressor.Embodiment 49. The method of Embodiment 48, wherein the light-controllable transcriptional repressor comprises a transcriptional repressor fused to a light-controllable domain.Embodiment 50. The method of Embodiment 49, wherein illumination of the cell with light at a specific wavelength or wavelength range induces the light-controllable transcriptionalWSGR Docket No. 60885-743.601repressor to dissociate from the constitutive promoter or a binding site close proximity to the constitutive promoter, thereby causing expression of the at least one viral protein.Embodiment 51. The method of Embodiment 49, wherein removing light or removing light at a specific wavelength or wavelength range from the cell induces the light-controllable transcriptional repressor to dissociate from the constitutive promoter or a binding site close proximity to the constitutive promoter, thereby causing expression of the at least one viral protein. Embodiment 52. The method of Embodiment 47, wherein the exogenous nucleic acid further comprises a blocking sequence downstream of the promoter which, when present, blocks expression of the at least one viral protein.Embodiment 53. The method of Embodiment 52, wherein the cell further comprises a nucleic acid sequence encoding a recombinase.Embodiment 54. The method of Embodiment 53, wherein the recombinase comprises a light-controllable recombinase.Embodiment 55. The method of Embodiment 54, wherein the light-controllable recombinase is a light-activatable recombinase.Embodiment 56. The method of Embodiment 55, wherein the light-activatable recombinase comprises a recombinase or a portion thereof fused to a light-activatable domain.Embodiment 57. The method of Embodiment 43, wherein the recombinase is a constitutively active recombinase.Embodiment 58. The method of Embodiment 47, wherein expression of the constitutively active recombinase is controllable by light.Embodiment 59. The method of any one of Embodiment 43 to Embodiment 58, wherein the blocking sequence is flanked by recombinase recognition sites that are recognized by the recombinase.Embodiment 60. The method of Embodiment 59, wherein the recombinase excises the blocking sequence thereby inducing expression of the at least one viral protein.Embodiment 61. The method of any one of Embodiment 1 to Embodiment 60, wherein the nucleic acid sequence encoding the at least one viral protein is codon-optimized.Embodiment 62. The method of any one of Embodiment 1 to Embodiment 61, wherein the cell further comprises a nucleic acid sequence encoding a viral genome.Embodiment 63. The method of Embodiment 62, wherein the viral genome comprises an adeno-associated virus (AAV) genome.Embodiment 64. The method of Embodiment 63, wherein the AAV genome comprises a recombinant AAV (rAAV) genome.WSGR Docket No. 60885-743.601Embodiment 65. The method of Embodiment 62, wherein the viral genome comprises a lentiviral genome.Embodiment 66. The method of any one of Embodiment 62 to Embodiment 65, wherein the viral genome comprises a gene expression cassette.Embodiment 67. The method of Embodiment 66, wherein the gene expression cassette comprises a transgene.Embodiment 68. The method of Embodiment 66 or Embodiment 67, wherein the gene expression cassette comprises a promoter.Embodiment 69. The method of Embodiment 68, wherein the promoter comprises a tissuespecific promoter.Embodiment 70. The method of Embodiment 68, wherein the promoter comprises a constitutive promoter.Embodiment 71. The method of Embodiment 68, wherein the promoter comprises an inducible promoter.Embodiment 72. The method of any one of Embodiment 66 to Embodiment 71, wherein the gene expression cassette comprises a nucleic acid sequence encoding a payload.Embodiment 73. The method of Embodiment 72, wherein the payload comprises a therapeutic payload.Embodiment 74. The method of Embodiment 70 or Embodiment 71, wherein the payload comprises a polypeptide.Embodiment 75. The method of any one of Embodiment 72 to Embodiment 74, wherein the payload comprises an RNA molecule.Embodiment 76. The method of any one of Embodiment 1 to Embodiment 75, wherein the at least one viral protein comprises a lentiviral protein.Embodiment 77. The method of any one of Embodiment 1 to Embodiment 75, wherein the at least one viral protein comprises an AAV protein.Embodiment 78. The method of Embodiment 77, wherein the AAV protein is selected from the group consisting of: an AAV1 protein, an AAV2 protein, an AAV3 protein, an AAV4 protein, an AAV5 protein, an AAV6 protein, an AAV7 protein, an AAV8 protein, an AAV9 protein, an AAV10 protein, an AAV11 protein, an AAV 12 protein, an AAV 13 protein, a variant thereof, and any combination thereof.Embodiment 79. The method of Embodiment 77 or Embodiment 78, wherein the AAV protein comprises an AAV Replicase (Rep) protein.WSGR Docket No. 60885-743.601Embodiment 80. The method of Embodiment 79, wherein the AAV Rep protein is selected from the group consisting of: an AAV Rep78, an AAV Rep68, an AAV Rep52, an AAV Rep 40 protein, and any combination thereof.Embodiment 81. The method of Embodiment 79 or Embodiment 80, wherein the at least one nucleic sequence does not comprise a functional internal pl9 promoter.Embodiment 82. The method of any one of Embodiment 77 to Embodiment 81, wherein the AAV protein comprises an AAV Capsid (Cap) protein.Embodiment 83. The method of Embodiment 82, wherein the AAV Cap protein comprises an engineered AAV Cap protein.Embodiment 84. The method of Embodiment 82, wherein the AAV Cap protein is selected from the group consisting of: a VP1 protein, a VP2 protein, a VP3 protein, a variant thereof, and any combination thereof.Embodiment 85. The method of any one of Embodiment 1 to Embodiment 75, wherein the at least one viral protein comprises at least one AAV helper protein.Embodiment 86. The method of Embodiment 85, wherein the at least one AAV helper protein is selected from the group consisting of: an adenovirus El A protein, an adenovirus E1B protein, an adenovirus E2B protein, an adenovirus E4orf6 protein, a herpesvirus protein, a cytomegalovirus (CMV) protein, a variant thereof, and any combination thereof.Embodiment 87. The method of any one of Embodiment 1 to Embodiment 86, wherein the cell further comprises a nucleic acid sequence encoding an adenoviral viral associated (VA) RNA. Embodiment 88. The method of any one of Embodiment 1 to Embodiment 87, wherein the cell produces a viral particle.Embodiment 89. The method of Embodiment 88, wherein the viral particle comprises a lentiviral particle.Embodiment 90. The method of Embodiment 88, wherein the cell produces an adeno-associated viral (AAV) particle.Embodiment 91. The method of Embodiment 90, wherein the AAV particle comprises a recombinant AAV (rAAV) particle.Embodiment 92. The method of Embodiment 91, wherein the rAAV particle is selected from the group consisting of: an rAAVl particle, an rAAV2 particle, an rAAV3 particle, an rAAV4 particle, an rAAV5 particle, an rAAV6 particle, an rAAV7 particle, an rAAV8 particle, an rAAV9 particle, an rAAVIO particle, an rAAVl 1 particle, an rAAV12 particle, an rAAV13 particle, a variant thereof, and any combination thereof.Embodiment 93. The method of any one of Embodiment 90 to Embodiment 92, wherein the AAV particle comprises an engineered AAV particle.WSGR Docket No. 60885-743.601Embodiment 94. The method of any one of Embodiment 1 to Embodiment 93, wherein the cell comprises a mammalian cell.Embodiment 95. The method of Embodiment 94, wherein the mammalian cell comprises a human cell.Embodiment 96. The method of Embodiment 95, wherein the human cell comprises a human embryonic kidney (EEK) 293 cell or a variant thereof.Embodiment 97. The method of Embodiment 96, wherein the HEK293 cell variant comprises a HEK293T cell.Embodiment 98. The method of Embodiment 97, wherein the HEK293 cell or variant thereof does not comprise an adenoviral El gene.Embodiment 99. The method of Embodiment 95, wherein the human cell comprises a HeLa cell.Embodiment 100. The method of any one of Embodiment 1 to Embodiment 93, wherein the cell comprises an insect cell.Embodiment 101. The method of Embodiment 100, wherein the insect cell comprises an Sf9 cell.Embodiment 102. The method of any one of Embodiment 1 to Embodiment 93, wherein the cell comprises abacterial cell.Embodiment 103. The method of any one of Embodiment 1 to Embodiment 102, further comprising controlling expression of the at least one viral protein using a closed loop control system.Embodiment 104. The method of Embodiment 103, wherein the closed loop control system comprises:(a) measuring the expression of the at least one viral protein, and(b) controlling the expression of the at least one viral protein using light by adjusting at least one light parameter.Embodiment 105. The method of Embodiment 104, wherein the at least one light parameter is selected from the group consisting of: wavelength, intensity, exposure time, and any combination thereof.Embodiment 106. The method of any one of Embodiment 1 to Embodiment 105, comprising controlling expression of two or more viral proteins with light, each of the two or more viral proteins controlled with light of a different wavelength or wavelength range.Embodiment 107. A cell comprising:at least one exogenous nucleic acid comprising at least one nucleic acid sequence operably linked to a promoter and encoding at least one viral protein; andWSGR Docket No. 60885-743.601at least one light-controllable transcriptional regulator or at least one nucleic acid sequence encoding thereof,wherein the at least one light-controllable transcriptional regulator is activatable, upon modulated exposure to a light, to induce expression of the at least one viral protein under the control of the promoter.Embodiment 108. The cell of Embodiment 107, wherein the at least one nucleic acid sequence does not comprise a functional internal promoter associated with expression of the at least one viral protein in a native viral context.Embodiment 109. The cell of Embodiment 108, wherein the at least one nucleic acid sequence does not comprise a gene encoding the functional internal promoter.Embodiment 110. The cell of any one of Embodiment 107 to Embodiment 109, wherein the at least one light-controllable transcriptional regulator is activatable, upon the modulated exposure to the light, to bind the promoter to induce the expression of the at least one viral protein.Embodiment 111. The cell of any one of Embodiment 107 to Embodiment 110, wherein the cell further comprises an additional nucleic acid sequence encoding an additional promoter and an additional viral protein, wherein the additional promoter is not inducible by the light, and wherein the at least one viral protein and the additional viral protein are different.Embodiment 112. The cell of Embodiment 111, wherein the cell comprises an additional exogenous nucleic acid comprising the additional nucleic acid sequence, wherein the at least one exogenous nucleic acid and the additional exogenous nucleic acid are different nucleic acid molecules.Embodiment 113. The cell of Embodiment 111, wherein the cell comprises an additional exogenous nucleic acid comprising the additional nucleic acid sequence, wherein the at least one exogenous nucleic acid and the additional exogenous nucleic acid are within a single nucleic acid molecule.Embodiment 114. The cell of any one of Embodiment 111 to Embodiment 113, wherein, wherein (i) the at least one viral protein comprises a helper protein and (ii) the additional viral protein comprises a replication protein, a capsid protein, or both.Embodiment 115. The cell of Embodiment 114, wherein the helper protein comprises an adenovirus helper protein comprising one or more members selected from the group consisting of E2A, E4, and L4.Embodiment 116. The cell of Embodiment 115, wherein the adenovirus helper protein comprises (i) E2A, (ii) E2A and E4, or (ii) E2A, E4, and L4.Embodiment 117. The cell of any one of Embodiment 111 to Embodiment 116, wherein the additional promoter comprises a functional viral promoter.WSGR Docket No. 60885-743.601Embodiment 118. The cell of Embodiment 117, wherein the functional viral promoter comprises p5, pl 9, or p40.Embodiment 119. The cell of any one of Embodiment 111 to Embodiment 113, wherein (i) the at least one viral protein comprises a replication protein and (ii) the additional viral protein comprises a helper protein, a capsid protein, or both.Embodiment 120. The method of Embodiment 119, wherein the at least one viral protein comprises a large Rep protein and a small Rep protein.Embodiment 121. The cell of Embodiment 120, wherein the at least one viral protein comprises Rep68 and Rep40 .Embodiment 122. The cell of Embodiment 120, wherein the at least one viral protein comprises Rep78 and Rep 52.Embodiment 123. The cell of Embodiment 120, wherein the additional nucleic acid sequence comprises a single nucleic acid molecule encoding both the large Rep protein and the small Rep protein.Embodiment 124. The cell of Embodiment 111, wherein at least a portion of the additional nucleic acid sequence encoding the additional viral protein is codon optimized.Embodiment 125. The cell of Embodiment 107 or Embodiment 124, wherein the cell is a producer cell.Embodiment 126. The cell of Embodiment 107 or Embodiment 124, wherein the cell is a packaging cell.Embodiment 127. The cell of any one of Embodiment 124 to Embodiment 126, wherein the promoter comprises an inducible promoter.Embodiment 128. The cell of Embodiment 127, wherein the at least one light-controllable transcriptional regulator comprises a light-controllable transcriptional activator.Embodiment 129. The cell of Embodiment 128, wherein the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain.Embodiment 130. The cell of Embodiment 129, wherein illumination of the cell with light at a specific wavelength or wavelength range induces the light-controllable transcriptional activator to bind to and activate the inducible promoter, thereby causing expression of the at least one viral protein.Embodiment 131. The cell of Embodiment 129, wherein removing light or light at a specific wavelength or wavelength range from the cell induces the light-controllable transcriptional activator to bind to and activate the inducible promoter, thereby causing expression of the at least one viral protein.WSGR Docket No. 60885-743.601Embodiment 132. The cell of any one of Embodiment 124 to Embodiment 126, wherein the promoter is a constitutive promoter.Embodiment 133. The cell of Embodiment 132, wherein the at least one light-controllable transcriptional regulator comprises a light-controllable transcriptional repressor.Embodiment 134. The cell of Embodiment 133, wherein the light-controllable transcriptional repressor comprises a transcriptional repressor fused to a light-controllable domain.Embodiment 135. The cell of Embodiment 134, wherein illumination of the cell with light at a specific wavelength or wavelength range induces the light-controllable transcriptional repressor to dissociate from the constitutive promote or a binding site close proximity to the constitutive promoter r, thereby causing expression of the at least one viral protein.Embodiment 136. The cell of Embodiment 134, wherein removing light or removing light at a specific wavelength or wavelength range from the cell induces the light-controllable transcriptional repressor to dissociate from the constitutive promoter or a binding site close proximity to the constitutive promoter, thereby causing expression of the at least one viral protein. Embodiment 137. The cell of Embodiment 132, wherein the exogenous nucleic acid further comprises a blocking sequence downstream of the promoter which, when present, blocks expression of the at least one viral protein.Embodiment 138. The cell of Embodiment 137, wherein the cell further comprises a nucleic acid sequence encoding a recombinase.Embodiment 139. The cell of Embodiment 138, wherein the recombinase comprises a light-controllable recombinase.Embodiment 140. The cell of Embodiment 139, wherein the light-controllable recombinase is a light-activatable recombinase.Embodiment 141. The cell of Embodiment 140, wherein the light-activatable recombinase comprises a recombinase or a portion thereof fused to a light-activatable domain.Embodiment 142. The cell of Embodiment 138, wherein the recombinase is a constitutively active recombinase.Embodiment 143. The cell of Embodiment 142, wherein expression of the constitutively active recombinase is controllable by light.Embodiment 144. The cell of any one of Embodiment 137 to Embodiment 143, wherein the blocking sequence is flanked by recombinase recognition sites that are recognized by the recombinase.Embodiment 145. The cell of Embodiment 144, wherein the recombinase excises the blocking sequence thereby inducing expression of the at least one viral protein.WSGR Docket No. 60885-743.601Embodiment 146. The cell of any one of Embodiment 107 to Embodiment 145, wherein the nucleic acid sequence encoding the at least one viral protein is codon-optimized.Embodiment 147. The cell of any one of Embodiment 107 to Embodiment 146, wherein the cell further comprises a nucleic acid sequence encoding a viral genome.Embodiment 148. The cell of Embodiment 147, wherein the viral genome comprises an adeno-associated virus (AAV) genome.Embodiment 149. The cell of Embodiment 148, wherein the AAV genome comprises a recombinant AAV (rAAV) genome.Embodiment 150. The cell of Embodiment 148, wherein the viral genome comprises a lentiviral genome.Embodiment 151. The cell of any one of Embodiment 147 to Embodiment 150, wherein the viral genome comprises a gene expression cassette.Embodiment 152. The cell of Embodiment 151, wherein the gene expression cassette comprises a transgene.Embodiment 153. The cell of Embodiment 151 or Embodiment 152, wherein the gene expression cassette comprises a promoter.Embodiment 154. The cell of Embodiment 153, wherein the promoter comprises a tissuespecific promoter.Embodiment 155. The cell of Embodiment 153, wherein the promoter comprises a constitutive promoter.Embodiment 156. The cell of Embodiment 153, wherein the promoter comprises an inducible promoter.Embodiment 157. The cell of any one of Embodiment 151 to Embodiment 156, wherein the gene expression cassette comprises a nucleic acid sequence encoding a payload.Embodiment 158. The cell of Embodiment 157, wherein the payload comprises a therapeutic payload.Embodiment 159. The cell of Embodiment 157 or Embodiment 158, wherein the payload comprises a polypeptide.Embodiment 160. The cell of Embodiment 157 or Embodiment 158, wherein the payload comprises an RNA molecule.Embodiment 161. The cell of any one of Embodiment 107 to Embodiment 160, wherein the at least one viral protein comprises a lentiviral protein.Embodiment 162. The cell of any one of Embodiment 107 to Embodiment 160, wherein the at least one viral protein comprises an AAV protein.WSGR Docket No. 60885-743.601Embodiment 163. The cell of Embodiment 162, wherein the AAV protein is selected from the group consisting of: an AAV1 protein, an AAV2 protein, an AAV3 protein, an AAV4 protein, an AAV5 protein, an AAV6 protein, an AAV7 protein, an AAV8 protein, an AAV9 protein, an AAV10 protein, an AAV11 protein, an AAV 12 protein, an AAV 13 protein, a variant thereof, and any combination thereof.Embodiment 164. The cell of Embodiment 162 or Embodiment 163, wherein the AAV protein comprises an AAV Replicase (Rep) protein.Embodiment 165. The cell of Embodiment 164, wherein the AAV Rep protein is selected from the group consisting of: an AAV Rep78, an AAV Rep68, an AAV Rep52, an AAV Rep 40 protein, and any combination thereof.Embodiment 166. The cell of Embodiment 164 or Embodiment 165, wherein the at least one nucleic sequence does not comprise a functional internal pl9 promoter.Embodiment 167. The cell of any one of Embodiment 162 to Embodiment 166, wherein the AAV protein comprises an AAV Capsid (Cap) protein.Embodiment 168. The cell of Embodiment 167, wherein the AAV Cap protein comprises an engineered AAV Cap protein.Embodiment 169. The cell of Embodiment 167 or Embodiment 168, wherein the AAV Cap protein is selected from the group consisting of: a VP1 protein, a VP2 protein, a VP3 protein, a variant thereof, and any combination thereof.Embodiment 170. The cell of any one of Embodiment 107 to Embodiment 160, wherein the at least one viral protein comprises at least one AAV helper protein.Embodiment 171. The cell of Embodiment 170, wherein the at least one AAV helper protein is selected from the group consisting of: an adenovirus El A protein, an adenovirus E1B protein, an adenovirus E2B protein, an adenovirus E4 protein, a herpesvirus protein, a cytomegalovirus (CMV) protein, a variant thereof, and any combination thereof.Embodiment 172. The cell of any one of Embodiment 107 to Embodiment 171, wherein the cell further comprises a nucleic acid sequence encoding an adenoviral viral associated (VA) RNA. Embodiment 173. The cell of any one of Embodiment 107 to Embodiment 172, wherein the cell produces a viral particle.Embodiment 174. The cell of Embodiment 173, wherein the viral particle comprises a lentiviral particle.Embodiment 175. The cell of Embodiment 173, wherein the viral particle comprises an adeno-associated viral (AAV) particle.Embodiment 176. The cell of Embodiment 175, wherein the AAV particle comprises a recombinant AAV (rAAV) particle.WSGR Docket No. 60885-743.601Embodiment 177. The cell of Embodiment 176, wherein the rAAV particle is selected from the group consisting of: an rAAVl particle, an rAAV2 particle, an rAAV3 particle, an rAAV4 particle, an rAAV5 particle, an rAAV6 particle, an rAAV7 particle, an rAAV8 particle, an rAAV9 particle, an rAAVIO particle, an rAAVl 1 particle, an rAAV12 particle, an rAAV13 particle, a variant thereof, and any combination thereof.Embodiment 178. The cell of any one of Embodiment 175 to Embodiment 177, wherein the AAV particle comprises an engineered AAV particle.Embodiment 179. The cell of any one of Embodiment 107 to Embodiment 178, wherein the cell comprises a mammalian cell.Embodiment 180. The cell of Embodiment 179, wherein the mammalian cell comprises a human cell.Embodiment 181. The cell of Embodiment 180, wherein the human cell comprises a human embryonic kidney (EEK) 293 cell or a variant thereof.Embodiment 182. The cell of Embodiment 181, wherein the HEK293 cell variant comprises a HEK293T cell.Embodiment 183. The cell of Embodiment 181, wherein the HEK293 cell or variant thereof does not comprise an adenoviral El gene.Embodiment 184. The cell of Embodiment 180, wherein the human cell comprises a HeLa cell.Embodiment 185. The cell of any one of Embodiment 107 to Embodiment 178, wherein the cell comprises an insect cell.Embodiment 186. The cell of Embodiment 185, wherein the insect cell comprises an Sf9 cell. Embodiment 187. The cell of any one of Embodiment 107 to Embodiment 178, wherein the cell comprises abacterial cell.Embodiment 188. A cell culture comprising a plurality of the cells of any one of Embodiment 107 to Embodiment 187; and a cell culture media.Embodiment 189. The cell culture of Embodiment 188, wherein the plurality of the cells is in suspension.Embodiment 190. A bioreactor comprising the cell culture of Embodiment 188 or Embodiment 189.Embodiment 191. The bioreactor of Embodiment 190, wherein the bioreactor has a total volume of at least 100 milliliters (mL).Embodiment 192. The bioreactor of Embodiment 190 or Embodiment 191, wherein the bioreactor further comprises at least one light source.WSGR Docket No. 60885-743.601Embodiment 193. The bioreactor of Embodiment 192, wherein the at least one light source comprises at least one light emitting diode (LED).Embodiment 194. The bioreactor of Embodiment 193, wherein the at least one LED comprises at least two different LEDs.Embodiment 195. The bioreactor of Embodiment 194, wherein the at least two different LEDs emit light at different wavelengths.Embodiment 196. The bioreactor of Embodiment 192, wherein the at least one light source comprises at least one laser.Embodiment 197. The bioreactor of Embodiment 192, wherein the at least one light source comprises at least one incandescent light source.Embodiment 198. The bioreactor of any one of Embodiment 190 to Embodiment 197, wherein the at least one light source is located inside the bioreactor, located on an interior surface of the bioreactor, or both.Embodiment 199. The bioreactor of any one of Embodiment 190 to Embodiment 198, wherein the at least one light source is located outside the bioreactor, on an exterior surface of the bioreactor, or both.Embodiment 200. The bioreactor of any one of Embodiment 190 to Embodiment 199, wherein the bioreactor comprises at least one wall or surface that is optically transparent.Embodiment 201. The bioreactor of any one of Embodiment 190 to Embodiment 200, further comprising a temperature source for controlling a temperature of the culture media.Embodiment 202. The bioreactor of any one of Embodiment 190 to Embodiment 201, further comprising an agitation source for agitating the culture media.Embodiment 203. The bioreactor of any one of Embodiment 190 to Embodiment 202, further comprising a controller for controlling illumination.

[0291] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of theWSGR Docket No. 60885-743.601invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No. 60885-743.601CLAIMSWHAT IS CLAIMED IS:

1. A method of producing at least one viral protein, the method comprising:(a) providing or obtaining a cell comprising at least one exogenous nucleic acid comprising at least one nucleic acid sequence operably linked to a promoter and encoding at least one viral protein, and(b) controlling expression of the at least one viral protein using light.

2. The method of claim 1, wherein (b) comprises controlling the expression of the at least one viral protein via a temporary modulated exposure of the cell to the light.

3. The method of claim 2, wherein the temporary modulated exposure is provided by supplying the light to the cell and subsequently removing the light from the cell.

4. The method of claim 2, wherein the temporary modulated exposure effects transient expression of the at least one viral protein in the cell.

5. The method of claim 2, wherein a duration of the temporary modulated exposure is between about 0.5 hours and 60 hours.

6. The method of claim 5, wherein the duration of the temporary modulated exposure is between about 6 hours and 60 hours.

7. The method of claim 5, wherein the duration of the temporary modulated exposure is between about 10 hours and 60 hours.

8. The method of claim 5, wherein the duration of the temporary modulated exposure is between about 24 hours and 60 hours.

9. The method of claim 2, wherein the temporary modulated exposure comprises a continuous temporary modulated exposure.

10. The method of claim 2, further comprising culturing the cell for a culture period to produce a virus particle.

11. The method of claim 10, wherein the temporary modulated exposure is initiated during the initial 50% of the culture period.

12. The method of claim 10, wherein the temporary modulated exposure is initiated during the initial 30% of the culture period.

13. The method of claim 10, wherein the temporary modulated exposure is initiated within the initial 36 hours of the culture period.

14. The method of claim 10, wherein the temporary modulated exposure is initiated within the initial 24 hours of the culture period.WSGR Docket No. 60885-743.60115. The method of claim 10, wherein a duration of the temporary modulated exposure is shorter than the culture period by at least about 10%, at least about 20%, or at least about 30%.

16. The method of claim 10, wherein the temporary modulated exposure of the cell to the light yields a production level of the virus particle that is greater than that produced via use of a comparable temporary modulated exposure to the light that is initiated at a later time point during the culture period than the temporary modulated exposure.

17. The method of claim 16, wherein the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at least about 10%, at least about 50%, at least about 100%, or at least about 200%.

18. The method of claim 16, wherein the production level of the virus particle via the temporary modulated exposure is greater than that via the comparable temporary modulated exposure by at least about 1-fold, at least about 2-fold, at least about 3 -fold, or at least about 5 -fold.

19. The method of claim 2, wherein the temporary modulated exposure of the cell to the light yields a production level of the virus particle that is greater than that produced by via use of a longer exposure to the light.

20. The method of claim 19, wherein the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at least about 10%, at least about 50%, at least about 100%, or at least about 200%.

21. The method of claim 19, wherein the production level of the virus particle via the temporary modulated exposure is greater than that via the longer exposure by at least about 0.5-fold, at least about 1-fold, or at least about 2-fold.

22. The method of claim 1, wherein the at least one nucleic acid sequence does not comprise a functional internal promoter associated with expression of the at least one viral protein in a native viral context.

23. The method of claim 22, wherein the at least one nucleic acid sequence does not comprise a gene encoding the functional internal promoter.

24. The method of claim 1, wherein the promoter of the at least one nucleic acid sequence is inducible by the light.

25. The method of claim 24, wherein the promoter is activated via a light controllable transcriptional regulator.

26. The method of claim 2, wherein the cell comprises an additional nucleic acid sequence encoding an additional promoter and an additional viral protein, wherein the additionalWSGR Docket No. 60885-743.601promoter is not inducible by the light, and wherein the at least one viral protein and the additional viral protein are different.

27. The method of claim 26, wherein the cell comprises an additional exogenous nucleic acid comprising the additional nucleic acid sequence, wherein the at least one exogenous nucleic acid and the additional exogenous nucleic acid are different nucleic acid molecules.

28. The method of claim 26, wherein the cell comprises an additional exogenous nucleic acid comprising the additional nucleic acid sequence, wherein the at least one exogenous nucleic acid and the additional exogenous nucleic acid are within a single nucleic acid molecule.

29. The method of claim 26, wherein (i) the at least one viral protein comprises a helper protein and (ii) the additional viral protein comprises a replication protein, a capsid protein, or both.

30. The method of claim 29, wherein the helper protein comprises an adenovirus helper protein comprising one or more members selected from the group consisting of E2A, E4, and L4.

31. The method of claim 30, wherein the adenovirus helper protein comprises (i) E2A, (ii) E2A and E4, or (ii) E2A, E4, and L4.

32. The method of claim 29, wherein the additional promoter comprises a functional viral promoter.

33. The method of claim 32, wherein the functional viral promoter comprises p5, pl9, or p40.

34. The method of claim 26, wherein (i) the at least one viral protein comprises a replication protein and (ii) the additional viral protein comprises a helper protein, a capsid protein, or both.

35. The method of claim 34, wherein the at least one viral protein comprises a large Rep protein and a small Rep protein.

36. The method of claim 35, wherein the at least one viral protein comprises Rep68 and Rep40.

37. The method of claim 35, wherein the at least one viral protein comprises Rep78 and Rep 52.

38. The method of claim 35, wherein the additional nucleic acid sequence comprises a nucleic acid molecule encoding the large Rep protein and a separate nucleic acid molecule encoding the small Rep protein.

39. The method of claim 26, wherein at least a portion of the additional nucleic acid sequence encoding the additional viral protein is codon optimized.

40. The method of claim 1, wherein the cell further comprises at least one nucleic acid sequence encoding at least one light-controllable transcriptional regulator.WSGR Docket No. 60885-743.60141. The method of claim 40, wherein the expression of the at least one viral protein is controllable by the at least one light-controllable transcriptional regulator.

42. The method of claim 41, wherein the promoter comprises an inducible promoter.

43. The method of claim 42, wherein the at least one light-controllable transcriptional regulator comprises a light-controllable transcriptional activator.

44. The method of claim 43, wherein the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain.

45. The method of claim 44, wherein illumination of the cell with light at a specific wavelength or wavelength range induces the light-controllable transcriptional activator to bind to and activate the inducible promoter or a binding domain thereof, thereby causing expression of the at least one viral protein.

46. The method of claim 1, wherein the at least one viral protein comprises a lentiviral protein.

47. The method of claim 1, wherein the at least one viral protein comprises an AAV protein.

48. The method of claim 1, wherein the cell comprises a mammalian cell.

49. The method of claim 48, wherein the mammalian cell comprises a human cell.

50. A cell comprising:at least one exogenous nucleic acid comprising at least one nucleic acid sequence operably linked to a promoter and encoding at least one viral protein; andat least one light-controllable transcriptional regulator or at least one nucleic acid sequence encoding thereof,wherein the at least one light-controllable transcriptional regulator is activatable, upon modulated exposure to a light, to induce expression of the at least one viral protein under the control of the promoter.

51. The cell of claim 50, wherein the at least one nucleic acid sequence does not comprise a functional internal promoter associated with expression of the at least one viral protein in a native viral context.

52. The cell of claim 51, wherein the at least one nucleic acid sequence does not comprise a gene encoding the functional internal promoter.

53. The cell of claim 50, wherein the at least one light-controllable transcriptional regulator is activatable, upon the modulated exposure to the light, to bind the promoter to induce the expression of the at least one viral protein.

54. The cell of claim 50, wherein the cell further comprises an additional nucleic acid sequence encoding an additional promoter and an additional viral protein, wherein the additional promoter is not inducible by the light, and wherein the at least one viral protein and the additional viral protein are different.WSGR Docket No. 60885-743.60155. The cell of claim 54, wherein the cell comprises an additional exogenous nucleic acid comprising the additional nucleic acid sequence, wherein the at least one exogenous nucleic acid and the additional exogenous nucleic acid are different nucleic acid molecules.

56. The cell of claim 54, wherein the cell comprises an additional exogenous nucleic acid comprising the additional nucleic acid sequence, wherein the at least one exogenous nucleic acid and the additional exogenous nucleic acid are within a single nucleic acid molecule.

57. The cell of claim 54, wherein, wherein (i) the at least one viral protein comprises a helper protein and (ii) the additional viral protein comprises a replication protein, a capsid protein, or both.

58. The cell of claim 57, wherein the helper protein comprises an adenovirus helper protein comprising one or more members selected from the group consisting of E2A, E4, and L4.

59. The cell of claim 58, wherein the adenovirus helper protein comprises (i) E2A, (ii) E2A and E4, or (ii) E2A, E4, and L4.

60. The cell of claim 54, wherein the additional promoter comprises a functional viral promoter.

61. The cell of claim 60, wherein the functional viral promoter comprises p5, pl 9, or p40.

62. The cell of claim 54, wherein (i) the at least one viral protein comprises a replication protein and (ii) the additional viral protein comprises a helper protein, a capsid protein, or both.

63. The method of claim 62, wherein the at least one viral protein comprises a large Rep protein and a small Rep protein.

64. The cell of claim 63, wherein the at least one viral protein comprises Rep68 and Rep40 .

65. The cell of claim 63, wherein the at least one viral protein comprises Rep78 and Rep 52.

66. The cell of claim 63, wherein the additional nucleic acid sequence comprises a single nucleic acid molecule encoding both the large Rep protein and the small Rep protein.

67. The cell of claim 54, wherein at least a portion of the additional nucleic acid sequence encoding the additional viral protein is codon optimized.

68. The cell of claim 50 or 67, wherein the cell is a producer cell.

69. The cell of claim 50 or 67, wherein the cell is a packaging cell.

70. The cell of any of claims 67-69, wherein the promoter comprises an inducible promoter.

71. The cell of claim 70, wherein the at least one light-controllable transcriptional regulator comprises a light-controllable transcriptional activator.WSGR Docket No. 60885-743.60172. The cell of claim 71, wherein the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain.

73. The cell of claim 72, wherein illumination of the cell with light at a specific wavelength or wavelength range induces the light-controllable transcriptional activator to bind to and activate the inducible promoter, thereby causing expression of the at least one viral protein.

74. The cell of any of claim 50, wherein the at least one viral protein comprises a lentiviral protein.

75. The cell of any of claim 50, wherein the at least one viral protein comprises an AAV protein.

76. The cell of claim 50, wherein the cell comprises a mammalian cell.

77. The cell of claim 76, wherein the mammalian cell comprises a human cell.

78. A cell culture comprising a plurality of the cell of any of claims 50 to 77; and a cell culture media.

79. The cell culture of claim 78, wherein the plurality of the cells is in suspension.

80. A bioreactor comprising the cell culture of claim 78 or 79.

81. The bioreactor of claim 80, wherein the bioreactor has a total volume of at least 100 milliliters (mL).

82. The bioreactor of claim 80 or 81, wherein the bioreactor further comprises at least one light source.

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