WO2023173105A2

WO2023173105A2 - Tetracycline inducer/repressor system and simplified viral particle production

Info

Publication number: WO2023173105A2
Application number: PCT/US2023/064165
Authority: WO
Inventors: Joseph J. HIGGINS; Feng Yao; Subhrangshu GUHATHAKURTA
Original assignee: Saliogen Therapeutics, Inc.; The Brigham And Women's Hospital, Inc.
Priority date: 2022-03-10
Filing date: 2023-03-10
Publication date: 2023-09-14
Also published as: WO2023173105A3

Abstract

The present disclosure describes engineered inducible tetracycline promoters for AAV production.

Description

TETRACYCLINE INDUCER/REPRESSOR SYSTEM AND SIMPLIFIED VIRAL PARTICLE PRODUCTION

FIELD

The present disclosure relates to non-leaky inducible tetracycline promoters and uses thereof, e.g., in making of viral particle packaging and producer cell lines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 63/318,598, filed March 10, 2022, and 63/335,860, filed April 28, 2022, all of which are incorporated by reference herein in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The Sequence Listing for this application is labeled “SAL-015PC_SequenceListing.XML”, which was created on March 7, 2023, and is 20,000 bytes in size.

BACKGROUND

The traditional method for Adeno-associated virus (AAV) vector production is a double plasmid transfection followed by a helper adenovirus (Ad) infection. The vector plasmid contains the transgene expression cassette that is flanked by the AAV inverted terminal repeats, while the packaging plasmid provides AAV Rep and Cap genes for vector DNA replication and packaging. Five Ad genes, i.e., those for E1 A, E1 B, E2A, E4, and VA RNA, are required for efficient AAV gene expression, DNA replication, and packaging. The key player of these five helper genes is the gene for E1 A, which the earliest gene product made during Ad infection. Ad E1A not only positively controls the expression of numerous Ad genes such as those for Ad E1 B, Ad E2A, and Ad E4, but also trans activates AAV Rep and Cap genes. AAV Rep and Cap genes are well known to be cytostatic and cytotoxic, making it extremely difficult to obtain a stable cell line from cells that constitutively express Ad E1A such as HEK293 cells. Silencing the AAV Rep and Cap genes, and Ad E2A, genes are crucial to the success of creating an inducible, stable AAV packaging cell line in HEK293 cells that constitutively express E1A.

AAV manufacturing is performed by using the HEK293 cell line and transient expression with a three-plasmid system (“triple transfection”). The three plasmids encode the various components needed to produce AAV, including a genome/transgene vector, a packaging plasmid providing the REP and CAP genes, and a helper plasmid that supplies the Ad5 genes E2A, E4, and VA. Two other adenovirus genes required for AAV replication, E1A and E1 B, are already expressed by HEK293. A major technical challenge with developing packaging cell lines is preventing cell death due to the cytotoxicity of the E4, E2A, and REP genes.

SUMMARY

Accordingly, the present disclosure describes, in part, placing certain genes in a single plasmid with the toxic REP and E2A genes under control of a tetracycline inducer/repressor system. Additionally, this disclosure describes, in part, producing packaging cell lines capable of regulated gene amplification combined tetracycline-controlled gene expression. In these cells, in embodiments, a tet-operator-containing P5 promoter sequence controls REP and CAP gene expression while a modified tet-operator-containing SV40 promoter controls Ad E2A expression as shown in FIG. 2. Because the REP and Ad E2A proteins are expressed only after exposure of Ad E1 A in HEK293 cells, without wishing to be bound by theory, this strategy avoids or reduces the toxicity of these proteins in producer cell lines. Producer cells can be generated from these packaging lines by the addition of a gene of interest vector flanked by AAV ITRs and linked to the SV40 origin, which will also amplify in the presence of doxycycline, excise, replicate, and be packaged into virions once REP and CAP are expressed.

In aspects, there is provided a composition comprising a recombinant tetracycline inducer/repressor system comprising at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, and a modified TATA box, wherein the tetracycline inducer/repressor system provides substantially non-leaky control of expression of a gene in the vector (e.g., one or more components of the donor DNA described herein).

In aspects, there is provided a composition comprising a recombinant tetracycline inducer/repressor system comprising at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, and a modified TATA box element and at least one tet operator (tetO) downstream of the TATA box, wherein the tetracycline inducer/repressor system provides substantially non-leaky control of expression of a gene in the vector (e.g., one or more components of the donor DNA described herein).

In aspects, there is provided a composition comprising a recombinant tetracycline inducer/repressor system comprising at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, and a modified TATA box element containing at least one tet operator (tetO) downstream of the TATA box, and tetracycline repressor, tetR, wherein the tetracycline inducer/repressor system provides substantially non-leaky control of expression of a gene in the vector (e.g., one or more components of the donor DNA described herein).

In aspects, there is provided a composition comprising a recombinant tetracycline inducer/repressor system comprising at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, and a modified TATA box element and at least one tet operator (tetO), wherein the tetracycline inducer/repressor system is suitable for use controlling transgene expression by tight regulation under repressed conditions.

In aspects, there is provided a composition comprising a recombinant tetracycline inducer/repressor system comprising at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, and a modified TATA box element and at least one tet operator (tetO) downstream of the TATA box, wherein the tetracycline inducer/repressor system is suitable for use controlling transgene expression by tight regulation under repressed conditions.

In aspects, there is provided a composition comprising a recombinant tetracycline inducer/repressor system comprising at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, and a modified TATA box element and at least one tet operator (tetO) wherein the tetracycline inducer/repressor system is suitable for use in a single or double vector expression system that provides substantially non-leaky control of expression of a gene in a vector (e.g., one or more components of the donor DNA described herein).

In aspects, there is provided a composition comprising a recombinant tetracycline inducer/repressor system comprising at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, and a modified TATA box element and at least one tet operator (tetO) downstream of the TATA box, wherein the tetracycline inducer/repressor system is suitable for use in a single or double vector expression system that provides substantially non-leaky control of expression of a gene in a vector (e.g., one or more components of the donor DNA described herein).

In aspects, there is provided a method of making a viral particle packaging cell line, the method comprising transfecting an E1 A+, E1 B+ cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, and a second nucleic acid encoding a donor DNA under control of a recombinant tetracycline inducer/repressor system and comprising terminal ends recognized by the enzyme, wherein the donor DNA comprises: inducible viral replication (REP) and Capsid (CAP) genes, one or more adenoviral auxiliary genes, optionally selected from E4, E2A, and VA of an AAV, and one or more insulators, optionally selected from HS4, D4Z4, and wherein the tetracycline inducer/repressor system comprises: at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, a modified TATA box element, and at least one tet operator, optionally downstream of the TATA box, to thereby result in a viral particle packaging cell line. In embodiments, the method further comprising converting the packaging cell line into a producer cell by contacting the packaging cell line with a third nucleic acid encoding a donor DNA comprising a transgene flanked by AAV inverted terminal repeats (ITRs) to result in a transfected cell that expresses the transgene in association with the viral particle. In embodiments, the second nucleic acid is substantially of the form as show in FIG. 2, or a functional equivalent thereof.

In aspects, there is provided a method of making a viral particle packaging and producer cell line, the method comprising transfecting an E1A, E1 B+ cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, and a second nucleic acid encoding a donor DNA under control of a recombinant tetracycline inducer/repressor system and comprising terminal ends recognized by the enzyme and a transgene disposed between the terminal end, wherein the donor DNA comprises inducible viral replication (REP) and Capsid (CAP) genes, one or more adenoviral auxiliary genes, optionally selected from E4, E2A, and VA of an AAV, and one or more insulators, optionally selected from HS4, D4Z4, and wherein the tetracycline inducer/repressor system comprises at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, a modified TATA box element, and at least one tet operator, optionally downstream of the TATA box, to thereby result in a transfected cell that expresses the transgene in association with a viral particle.

In embodiments, the tetracycline inducer/repressor provides substantially non-leaky control of expression. In embodiments, the tetracycline inducer/repressor is induced by doxycycline. In embodiments, the tetracycline inducer/repressor provides substantially non-leaky control of expression of one or more of E4, E2A, and REP.

In embodiments, leakiness is assessed by measuring a transcription level at the promoter inactive state. In embodiments, a ratio of expression in the promoter inactive state to expression in the promoter active state to is at least about 1 :100, or at least about 1 :500, or at least about 1 :1000, or at least about 1 :5000, or at least about 1 :10000.

In embodiments, the tetracycline inducer/repressor further comprises a tet operator sequence positioned at least about 4 to about 12 nucleotides, or at least about to about 12 nucleotides, or at least about 4 to about 6 nucleotides 3' or downstream to the modified TATA box element. In embodiments, the modified TATA box element has the nucleotide sequence of TATATAA or TAT AT AG. In embodiments, the tetracycline inducer/repressor further comprises one or more enhancer elements. In embodiments, the enhancer element comprises one or more cis-acting elements, optionally selected from an Sp 1 binding site, GC rich sequence, GCGGAAC motif, TAATGARAT element, AP1 binding site, and CCAAT box element.

In embodiments, the enzyme capable of performing targeted genomic integration is a mobile element enzyme. In embodiments, the mobile element enzyme has the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least about 80% to at least about 99% identity thereto. In embodiments, the mobile element enzyme comprises an amino acid other than serine at the position corresponding to position 2 of SEQ ID NO: 7, optionally a non-polar aliphatic amino acid, optionally a non-polar aliphatic amino acid optionally selected from G, A, V, L, I and P, optionally A. In embodiments, the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 7. In embodiments, the enzyme has one or more mutations which confer hyperactivity. In embodiments, the enzyme comprises a targeting element, e.g., without limitation, one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, a transcription activator-like effector (TALE), or a Zinc finger (ZF).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a non-limiting schematic of the function of the tet operator-containing mammalian cells promoter. In the top panel, gene expression is off. “TBP” is a TATA-binding protein and “tetR” is a Tet repressor protein. In the bottom panel, gene expression is on.

FIG. 2 depicts the DNA element that is integrated into the HEK293 genome by a transposase or other mobile element. The construct contains a replication backbone (e.g., plasmid or miniplasmid) with tetracycline operator (tetO)-containing promoters driving the AAV REP/CAP genes (enhanced tetO; SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4), Bicistronic Adenovirus E2A gene-IRES-AdV E40rf6 (tetO- containing SV40 early major promoter, SEQ ID NO: 5), VA (HSV-2 ICP4; SEQ ID NO: 6) gene, and “Saliogase” in this figure refers to the mobile element enzyme of SEQ ID NO: 7.

FIG. 3 depicts 293R-22 cells, a HEK293 cells-derived cell line that stably expressing tetracycline repressor, tetR, transfected with a plasmid with EGFP underthe control of the tetO-containing hCMV IE promoter. 293R- 22 cells were transfected with 0.1 pg of pCDNA4TO-EGFP and 1.4 pg of pMP4 in either the absence or presence of doxycycline (Dox). Photos were taken 48 h post-transfection. The results demonstrate, inter alia, that GFP expression from the tetO-containing hCMV IE promoter can be tightly suppressed in the absence of doxycycline. In contrast, high levels of GFP expression are detected in the presence of doxycycline.

FIGs. 4A-4C depict transient expression of EGFP under the control of the enhanced tetO promoter (P5TO) in Vero, VOR-124 cells, and 293R-22 cells. VOR-124 cells and CCL-81 Vero cells were transfected with 0.25 pg of pP5TO-EGFP and 1 .25 pg of pMP4 (FIG. 4A); and 293R-22 cells were transfected with 0.5pg of pP5TO- EGFP and 1 pg of carrier DNA (FIG. 4B and FIG. 4C). Cells are cultured in the presence of 0.5 pg/ml of doxycycline (Dox). The results in FIG. 4A show that levels of EGFP expression from the P5TO promoter are significantly higher in V0R-124 cells than in CCL-81 Vero cells. VOR-124 is a CCL-81 Vero cell-derived stable cell line. V0R-124 expresses tetracycline repressor, tetR, and the HSV-1 major regulatory protein ICPO. ICPO is a potent transactivator that can activate gene expression in a promoter-sequence independent manner. The results indicate, inter alia, that the activity of the P5TO promoter can be efficiently enhanced by ICPO, which is stably expressed in VOR-124 cells. The results in FIG. 4B and FIG. 4C further demonstrate inducible eGFP expression under regulatable AAV P5-modified promoters (Switch 1-5) in HEK293R-22 Cells with or without doxycycline. C is a control plasmid where EGFP is under regulation of inducible CMV promoter in pCDNA4TO-EGFP. In FIG. 4B and FIG. 4C, for each sample, the left bar is “+” and the right bar is

FIGs. 5A-5B depict plasmid vectors used for transfection. FIG. 5A shows a complete AAV capsid producer donor element. This 13.2 kb DNA element contains two ITRs and two insulators (HS4, D4Z4) that flank components required for AAV production. The AAV2 REP/CAP gene is driven by a tetracycline-inducible promoter (enhanced tetO), and the bicistronic adenovirus 5 E2A-IRES-E40rf6 gene is driven by an inducible tetO SV40 promoter. “Saliogase” in this figure refers to the mobile element enzyme of SEQ ID NO: 7. FIG. 5B shows the 6.2 kb AAV2 CAG-GFP transgene flanked by AAV ITR. “Saliogase” in this figure refers to the mobile element enzyme of SEQ ID NO: 7.

FIGs. 6A-6D depict EGFP visualization hours after transduction of HEK-293 or U2OS cells with unconcentrated supernatant collected after 3-cycles of freeze-thaw from cells transfected with complete AAV capsid producer donor element with tetracycline inducible promoters and AAV2 ITR CMV-GFP transgene. FIG. 6A and FIG. 6C show transduction with AAV-EGFP/DOX+ supernatant. FIG. 6B and FIG. 6D show transduction of AAV-EGFP/DOX- supernatant. FIG. 6A and FIG. 6B are transduced HEK-293 cells visualized at 72 hours. FIG. 6C and FIG. 6D are transduced U-2 OS cells visualized at 92 hours. DETAILED DESCRIPTION

In aspects, there is provided a composition comprising a recombinant tetracycline inducer/repressor system comprising at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, and a modified TATA box element and at least one tet operator (tetO), wherein the tetracycline inducer/repressor system provides substantially non-leaky control of expression of a gene in the vector (e.g., one or more components of the donor DNA described herein).

In aspects, there is provided a composition comprising a recombinant tetracycline inducer/repressor system comprising at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, and a modified TATA box element and at least one tet operator, wherein the tetracycline inducer/repressor system is suitable for use in a single or double vector expression system that provides substantially non- leaky control of expression of a gene in a vector e.g., one or more components of the donor DNA described herein).

In aspects, there is provided a method of making a viral particle packaging cell line, the method comprising transfecting an E1A+, E1 B+ cell with a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, and a second nucleic acid encoding a donor DNA under control of a recombinant tetracycline inducer/repressor system and comprising terminal ends recognized by the enzyme, wherein the donor DNA comprises: inducible viral replication (REP) and Capsid (CAP) genes, one or more adenoviral auxiliary genes, optionally selected from E4, E2A, and VA of an AAV, and one or more insulators, optionally selected from HS4, D4Z4, and wherein the tetracycline inducer/repressor system comprises: at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof, a modified TATA box element, and at least one tet operator, optionally downstream of the TATA box, to thereby result in a viral particle packaging cell line. In embodiments, the method further comprising converting the packaging cell line into a producer cell by contacting the packaging cell line with a third nucleic acid encoding a donor DNA comprising a transgene flanked by AAV inverted terminal repeats (ITRs) to result in a transfected cell that expresses the transgene in association with the viral particle. In embodiments, the second nucleic acid is substantially of the form as show in FIG. 2, or a functional equivalent thereof.

FIG. 1 is a non-limiting schematic of the function of the tet operator-containing mammalian cells promoter. In the top panel, TBP is prevented from interaction with the TATA element and tetR interacts with tetO and gene expression is off. In the bottom panel, tetracycline is introduced, and it interacts with tetR, TBP interacts with the TATA element, and gene expression is on.

Accordingly, in embodiments, the present compositions provide for tetO-comprising promoters that suppress or ablate gene expression in the presence of tetR and/or in the absence of tetracycline, or an analog thereof, or doxycycline, or an analog thereof. In embodiments, the present compositions provide for tetO-comprising promoters that allow for the release of tetR from the tetO-comprising promoter in the presence of tetracycline, or an analog thereof, or doxycycline, or an analog thereof. In embodiments, the present compositions provide for tetO-comprising promoters that allow for gene expression in the presence of tetR and/or tetracycline, or an analog thereof, or doxycycline, or an analog thereof.

Reduction or Ablation of Promoter Leakiness

Regulation of transgene expression is crucial for recombinant protein production, especially in the context of mammalian cells. This is often accomplished using inducible promoters that respond to stimuli to lead to transgene expression. However, inducible promoters can demonstrate insufficient levels of production and cause deleterious side effects. As an alternative to inducible promoters, attempts have been made to control mammalian gene expression using prokaryotic regulatory elements, e.g., based upon interactions between prokaryotic operators and repressor proteins to target eukaryotic transcription modulators to specific sites within a host cell genome or to directly inhibit gene expression using the prokaryotic repressor. However, in practice, these systems rarely demonstrate a binary “on/off” phenotype. Instead, it is common for such promoters to demonstrate a degree of leakiness. Specifically, even in the “off” or inactive state or noninduced state, the mammalian cell may express the transgene. In embodiments, the tetracycline inducer/repressor provides non-leaky control of expression. In embodiments, the tetracycline inducer/repressor provides non-leaky control of expression, e.g., of a component of the donor DNA as described herein, e.g., one or more of E4, E2A, and REP.

In embodiments, the tetracycline inducer/repressor provides substantially non-leaky control of expression. In embodiments, the tetracycline inducer/repressor provides substantially non-leaky control of expression, e.g., of a gene, e.g., of a component of the donor DNA as described herein.

In embodiments, the tetracycline inducer/repressor provides substantially non-leaky control of expression of one or more of REP, CAP, E1A, E1 B, E4, E2A, and VA. In embodiments, the tetracycline inducer/repressor provides substantially non-leaky control of expression of one or more of E4, E2A, and REP.

In embodiments, the tetracycline inducer/repressor provides substantially non-leaky control of expression of one or more of components of the donor DNA, or two or more of components of the donor DNA, or three or more of components of the donor DNA, or four or more of components of the donor DNA, or five or more of components of the donor DNA.

In embodiments, the tetracycline inducer/repressor is induced by tetracycline, or an analog thereof.

In embodiments, the tetracycline inducer/repressor is induced by doxycycline, or an analog thereof. In embodiments, the tetracycline inducer/repressor is induced by doxycycline hyclate and/or doxycycline monohydrate.

In embodiments, the leakiness is assessed by measuring a transcription level at the promoter “off” or inactive state or noninduced state. In embodiments, the leakiness is assessed by measuring a transcription level at the promoter “on” or active state or induced state and “off’ or inactive state or noninduced state and comparing the same.

In embodiments, the leakiness is assessed by measuring a transcription level in the absence of doxycycline, or an analog thereof. In embodiments, the leakiness is assessed by measuring a transcription level in the presence of doxycycline, or an analog thereof, and the absence of doxycycline, or an analog thereof, and comparing the same.

In embodiments, the ratio of expression in the promoter “off” or inactive state or noninduced state to expression in the promoter “on” or active state or induced state to is at least about 1 :100, or at least about 1 : 500, or at least about 1 : 1000, or at least about 1 : 5000, or at least about 1 : 10000. In embodiments, the ratio of expression in the doxycycline, or an analog thereof, absent state to expression in the doxycycline, or an analog thereof, present state is at least about 1 :100, or at least about 1 :500, or at least about 1 :1000, or at least about 1 :5000, or at least about 1:10000.

In embodiments, the expression is less leaky than that observed in an equivalent tetracycline inducer/repressor system having a strong promoter, instead of P5 promoter or SV40 early promoter. In embodiments, the strong promoter is the human cytomegalovirus (CMV) immediate-early promoter. In embodiments, the expression is less leaky than that observed in the construct of U.S. Patent No. 5,972,650.

Gene expression, and accordingly leakiness, can be measured using techniques know in the art. For example, in embodiments, low-to-mid-plex techniques, including but not limited to reporter gene assays, Northern blot, fluorescent in situ hybridization (FISH), and reverse transcription PCR (RT-PCR) are used. In embodiments, gene expression, and accordingly leakiness, is measured using, for example, higher-plex techniques, including but not limited, serial analysis of gene expression (SAGE), Tiling array, RNA-Seq/whole transcriptome shotgun sequencing (WTSS), high-throughput sequencing, multiplex PCR, multiplex ligationdependent probe amplification (MLPA), and Luminex/XMAP. A person skilled in the art will appreciate that a number of methods can be used to detect or quantify the level of gene expression, and accordingly leakiness, within a sample, including arrays, such as microarrays, RT-PCR (including quantitative PCR), nuclease protection assays and Northern blot analyses. In embodiments, gene expression, and accordingly leakiness, is assessed using a reporter gene under the control of the present tetracycline inducer/repressor, e.g., with or without doxycycline, or an analog thereof. For example, in embodiments, a fluorescent protein visual reporter is used. Non limiting examples of fluorescent protein visual reporters include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal , GFPuv, Sapphire, T- sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl , AsRed2, eqFP611 , mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein.

Stnjcture of the Tetracycline I nducer/Reoressor System In embodiments, the tetracycline inducer/repressor comprises a tet operator sequence positioned at least about 4 to about 10 nucleotides 3' or downstream to the modified TATA box element, optionally about 4 nucleotides, or about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides 3' or downstream to the modified TATA box element. In embodiments, the tetracycline inducer/repressor comprises a tet operator sequence positioned at least about 7 to about 12 nucleotides 3' or downstream to the modified TATA box element. In embodiments, the tetracycline inducer/repressor comprises a tet operator sequence positioned at least about 4 to about 6 nucleotides 3' or downstream to the modified TATA box element.

In embodiments, the tet operator is downstream of the TATA box, e.g., about 4 nucleotides to about 12 nucleotides, or about 6 nucleotides to about 10 nucleotides, or about 6 nucleotides to about 8 nucleotides, or about 8 nucleotides to about 10 nucleotides (e.g., about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides) 3' or downstream of the last nucleotide of the modified TATA box. In embodiments, the tet operator is downstream of the TATA box about 4 nucleotides to about 6 nucleotides 3' or downstream of the last nucleotide of the modified TATA box.

In embodiments, the tetracycline inducer/repressor further comprises a gene 3' or downstream to the tet operator sequence, the gene optionally being one or more components of the donor DNA, or one or more of one or more of REP, CAP, E1A, E1 B, E4, E2A, and VA, or one or more of E4, E2A, and REP. In embodiments, the at least one of a P5 promoter or SV40 early promoter is 5' or upstream of the modified TATA box element. In embodiments, the at least one of a P5 promoter or SV40 early promoter is 5’ or upstream of the modified TATA box element; the modified TATA box element is 5' or upstream of the one or more tet operator sequences; and/or the one or more tet operator sequences is 5' or upstream of one or more components of the donor DNA, or one or more of one or more of REP, CAP, E1A, E1 B, E4, E2A, and VA, or one or more of E4, E2A, and REP.

In embodiments, the modified TATA box element has the nucleotide sequence of TATATAA or TATATAG. In embodiments, the modified TATA box element has the nucleotide sequence of aggcgccggg (SEQ ID NO: 16), or a functional variant thereof, disposed 5’ therefrom. In embodiments, the modified TATA box element does not have the nucleotide sequence of TATTTAA, or TATTTAT, or TAATTTTTTT (SEQ ID NO: 17). In embodiments, the tetracycline inducer/repressor further comprises one or more enhancer elements. In embodiments, the promoter in tetracycline inducer/repressor system further comprises one or more enhancer elements. In embodiments, the enhancer element comprises one or more cis-acting elements, optionally selected from an Sp 1 binding site, GC rich sequence, GCGGAAC motif, TAATGARAT element, AP1 binding site, and CCAAT box element. In embodiments, the enhancer element comprises about 1 to about 5 Sp1 binding sites, optionally about 1 , or about 2, or about 3, or about 4, or about 5 Sp1 binding sites. In embodiments, the enhancer element comprises about 1 or about 2 GC rich sequences. In embodiments, the enhancer elements are derived from an HSV-1 ICP4 promoter. The sequence of the ICP4 promoter is known in the art (e.g., Perry, et al., J. Gen. Virol. 67:2365-2380 (1986); McGeoch et al., J. Gen. Virol. 72:3057-3075 (1991); McGeoch et al., Nucl. Acid Res. 14:1727-1745 (1986); Yao and Schaffer, J of Virol. 69:6249-6258 (1995), the entire contents of which are incorporated by reference in their entireties). In embodiments, the enhancer element comprises the nucleotide sequence of SEQ ID NO: 8, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto:

GC GGAACGGAAG CGGAAACCGC CGGATCGGGC GGTAATGAGA TGCCATGCGG GGCGGGGCGC GGACCCACCC GCCCTCGCGC CCCGCCCATG GCAGATGGCG CGGATGGGCG GGGCCGGGGG TTCGACCAAC GGGCC ( SEQ ID NO : 8 ) .

In embodiments, the enhancer element is 5' or upstream of the P5 promoter or SV40 early promoter.

In embodiments, the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 1 , or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. In embodiments, certain fragments of SEQ ID NO: 1 are indicated below.

In embodiments, a tet-operator-containing P5 promoter is provided. In embodiments, the tet-operator- containing P5 promoter comprises the nucleotide sequence of SEQ ID NO: 9, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

In embodiments, an enhancer element of HSV-1 ICP4 promoter is provided. In embodiments, the enhancer element of HSV-1 ICP4 promoter comprises the nucleotide sequence of SEQ ID NO: 8, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

SEQ ID NO: 1. Nucleotide sequence of enhanced tetO promoter

The tet-operator-containing P5 promoter sequence (italics, nucleotides 146-300, SEQ ID NO: 9 below) with YY1 +1 element (underlined bold, nucleotides 291-300, CTCCATTTT) and modified TATA box element (underlined italics, nucleotides 211-217, TATATAA), insertion of enhancer element of HSV-1 ICP4 promoter (bold, nucleotides 9-145, SEQ ID NO: 8 as listed above and below). The sequence is flanked by 5’-Pacl site (ttaattaa) and a 3’-Notl site (gcggccgc).

308 bp DNA

ORIGIN

1 TTAATTAAGC GGAACGGAAG CGGAAACCGC CGGATCGGGC GGTAATGAGA TGCCATGCGG

61 GGCGGGGCGC GGACCCACCC GCCCTCGCGC CCCGCCCATG GCAGATGGCG CGGATGGGCG

121 GGGCCGGGGG TTCGACCAAC GGGCCGGTCC TGTATTAGAG GTCACGTGAG TGTTTTGCGA

181 CATTTTGCGA CACCATGTGG TCACGCTGGG TATATAAGCA ACCGGTCTCC CTATCAGTGA

241 TAGAGATCTC CCTATCAGTG ATAGAGATCG GCCCGAGTGA GCACGCAGGG TCTCCATTTT

301 GCGGCCGC

SEQ ID NO: 9

GGTCC TGTATTAGAG GTCACGTGAG TGTTTTGCGA CATTTTGCGA CACCATGTGG TCACGCTGGG TATATAAGCA ACCGGTCTCC CTATCAGTGA TAGAGATCTC CCTATCAGTG ATAGAGATCG GCCCGAGTGA GCACGCAGGG T

SEQ ID NO: 8 GC GGAACGGAAG CGGAAACCGC CGGATCGGGC GGTAATGAGA TGCCATGCGG GGCGGGGCGC GGACCCACCC GCCCTCGCGC CCCGCCCATG GCAGATGGCG CGGATGGGCG GGGCCGGGGG TTCGACCAAC GGGCC

In embodiments, the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 2, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. In embodiments, certain fragments of SEQ ID NO: 2 are indicated below.

In embodiments, an enhanced tet-operator-containing P5 promoter is provided. In embodiments, the enhanced tet-operator-containing P5 promoter comprises the nucleotide sequence of SEQ ID NO: 10, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

In embodiments, a cis-acting elements of the HCMV IE promoter is provided. In embodiments, the cis-acting elements of the HCMV IE promoter comprise the nucleotide sequence of SEQ ID NO: 11 , or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

SEQ ID NO: 2. Nucleotide sequence enhanced tetO promoter-1

The enhanced tet-operator-containing P5 promoter sequence (italics, nucleotides 150-299, SEQ ID NO: 10 below) with insertion of proximal cis-acting elements of the HCMV IE promoter (bold, nucleotides 9-149, SEQ ID NO: 11 below) atthe 5’ end of P5 promoter with the modified TATA element (underlined italics, nucleotides 215-221 , TAT ATAA) without YY1 +1 element. The sequence is flanked by 5’-Pacl site (ttaattaa) and a 3’-Notl site (gcggccgc).

307 bp DNA

ORIGIN

1 TTAATTAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT 61 GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC

121 AACTCCGCCC CATTGACGCA AATGGGCGGG GTCCTGTATT AGAGGTCACG TGAGTGTTTT 181 GCGACATTTT GCGACACCAT GTGGTCACGC TGGGTATATA AGCAACCGGT CTCCCTATCA 241 GTGATAGAGA TCTCCCTATC AGTGATAGAG ATCGGCCCGA GTGAGCACGC AGGGTCTCCG 301 CGGCCGC

SEQ ID NO: 10

G GTCCTGTATT AGAGGTCACG TGAGTGTTTT GCGACATTTT GCGACACCAT GTGGTCACGC TGGGTATATA AGCAACCGGT CTCCCTATCA GTGATAGAGA TCTCCCTATC AGTGATAGAG ATCGGCCCGA GTGAGCACGC AGGGTCTCCG

SEQ ID NO: 11

TG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGG

In embodiments, the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 3, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. In embodiments, certain fragments of SEQ ID NO: 3 are indicated below.

In embodiments, another enhanced tet-operator-containing P5 promoter is provided. In embodiments, the enhanced tet-operator-containing P5 promoter comprises the nucleotide sequence of SEQ ID NO: 12, or a functional variant or fragment thereof, or sequence having at least about 70%, or least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

In embodiments, another cis-acting elements of the HCMV IE promoter is provided. In embodiments, the cisacting elements of the HCMV IE promoter comprise the nucleotide sequence of SEQ ID NO: 13, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

SEQ ID NO: 3. Nucleotide sequence enhanced tetO promoter-2

The enhanced tet-operator-containing P5 promoter sequence (italics, nucleotides 168-239, SEQ ID NO: 12 below) with insertion of cis-acting elements of the HCMV IE promoter (bold, nucleotides 9-167, SEQ ID NO: 13 below) with an additional copy of 19 bp repeat (bold underlined, nucleotides 9-27, CCCCATTGACGTCAATGGG, SEQ ID NO: 18) at the 5’ end of P5 promoter with the modified TATA element (underlined italics, nucleotides 233-239, tatataa) without the YY1 +1 element. The sequence is flanked by 5’- Pacl site (ttaattaa) and a 3’-Notl site (gcggccgc).

325 bp DNA

ORIGIN

1 TTAATTAACC CCATTGACGT CAATGGGCGG CGTGGATAGC GGTTTGACTC ACGGGGATTT 61 CCAAGTCTCC ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC 121 TTTCCAAAAT GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGggt cctgta t tag

181 aggtcacgtg agtgt t ttgc gaca t t t tgc gacacca tgt ggtcacgctg ggta ta taaG

241 CAACCGGTCT CCCTATCAGT GATAGAGATC TCCCTATCAG TGATAGAGAT CGgcccgagt

301 gagcacgcag ggtctccGCG GCCGC

SEQ ID NO: 12 ggt cctgta t tag aggtcacgtg agtgtt t tgc gacat t ttgc gacacca tgt ggtcacgctg ggta ta taa

SEQ ID NO: 13 CC CCATTGACGT CAATGGGCGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGG In embodiments, the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 4, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. In embodiments, certain fragments of SEQ ID NO: 4 are indicated below.

In embodiments, an another enhanced tet-operator-containing P5 promoter is provided. In embodiments, the enhanced tet-operator-containing P5 promoter comprises the nucleotide sequence of SEQ ID NO: 14, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

SEQ ID NO: 4. Nucleotide sequence enhanced tetO promoter-3.

The enhanced P5TO promoter sequence (italics, nucleotides 146-237, SEQ ID NO: 14 below) with insertion of the enhancer element of HSV-1 ICP4 promoter at the 5’ end of P5 promoter (bold, nucleotides 9-145, SEQ ID NO: 8 below), an additional copy of the R1-R2 repeat of the native P5 promoter (bold underlined italics, nucleotides 173-192, ttttgcgacattttgcgaca, SEQ ID NO: 15) and the modified TATA element (underlined italics, nucleotides 231-237, tatataa) without the YY1 +1 element. The sequence is flanked by 5’-Pacl site (ttaattaa) and a 3’-Notl site (gcggccgc) 323 bp DNA

ORIGIN

1 TTAATTAAGC GGAACGGAAG CGGAAACCGC CGGATCGGGC GGTAATGAGA TGCCATGCGG

61 GGCGGGGCGC GGACCCACCC GCCCTCGCGC CCCGCCCATG GCAGATGGCG CGGATGGGCG

121 GGGCCGGGGG TTCGACCAAC GGGCCggtcc tgta t tagag gtcacgtgag tgttttgcga

181 cattttgaga cat tt tgcga ca t tt tgcga cacca tgtgg tcacgctggg ta ta taaGCA

241 ACCGGTCTCC CTATCAGTGA TAGAGATCTC CCTATCAGTG ATAGAGATCG gcccgagtga

301 gcacgcaggg tctccGCGGC CGC

SEQ ID NO: 14 ggtcc tgta t tagag gtcacgtgag cacca tgtgg tcacgctggg ta ta taa

SEQ ID NO: 8

GC GGAACGGAAG CGGAAACCGC CGGATCGGGC GGTAATGAGA TGCCATGCGG GGCGGGGCGC GGACCCACCC GCCCTCGCGC CCCGCCCATG GCAGATGGCG CGGATGGGCG GGGCCGGGGG TTCGACCAAC GGGCC

In embodiments, the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 5, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. In embodiments, certain fragments of SEQ ID NO: 5 are indicated below.

SEQ ID NO: 5. Nucleotide sequence of tetO SV40 early major promoter

LOCUS Modified SV40/TO promoter. The normal TAATTTTTTT (SEQ ID NO: 17) element immediate upstream of the SV40 TATA box (underlined, nucleotides 252-258, tatataa) is replaced with aggcgccggg

(SEQ ID NO: 16) element (bold, nucleotides 242-251).

340 bp DNA

ORIGIN

1 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt

61 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca

121 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta

181 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga

241 caggcgccgg gtatataagc agagctctcc ctatcagtga tagagatctc cctatcagtg

301 atagagatcg agctcgcgtg tgcgaggccg aggccgcctcg

In embodiments, a modified HSV-2 ICP4 promoter is provided. In embodiments, the modified HSV-2 ICP4 promoter comprises the nucleotide sequence of SEQ ID NO: 6, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. In embodiments, certain fragments of SEQ ID NO: 6 are indicated below.

SEQ ID NO: 6. Nucleotide sequence of modified HSV-2 ICP4 promoter

LOCUS HSV-2 1 CP4 promoter 366 bp DNA

ORIGIN

1 gggccggcgg gggccaacgg gagcgcgggg ccggcatctc attaccacga acccggaagg

61 gcaggggagc gagcccgccc gcgacgaggg tctcattagc atcgcgggcg gaagcggaag

121 ccgcccgcgc cgggcgctaa tgagatgccg cgcgggcgga gcggcggcgg cgcgaccaac

181 gggccgccgc cacggacgcg gacgcgcggg cgtcggggcg gggccgcgca taatgcggtt

241 ccacctgggg gcggaacccc ggcgagccgg ggcgcggcgg cgtcgatcgc tcctcctccg

301 cgtcctcctc ctttcccccc gccccgcgcg ccccgaggac tatatgagcc aggcgacggg

361 gcgatc

In embodiments, there is provided a cell comprising the tetracycline inducer/repressor described herein. In embodiments, the cell is selected from a human embryonic kidney (HEK293), Chinese hamster ovary (CHO) E1 A, E1 B+ engineered CHO-K1 , Spodoptera frugiperda (Sf9) cell line, baby hamster kidney (BHK), and Vero cell. In embodiments, the HEK293 cell stably expresses tetR. In embodiments, the cell is selected from a Vero cell-derived cell line. In embodiments, the Vero cell-derived cell line is VOR-124. In embodiments, the VOR-124 is highly stable at expressing both tetracycline Repressor (tetR) and ICPO protein. The types of cells to be used are described, for example, in US Publication No. 2020/0172928, which is hereby incorporated by reference in its entirety.

In embodiments, the tetracycline inducer/repressor system comprises one or more genes encoding tet repressor (tetR), or a codon-optimized tetR.

In embodiments, the tetracycline inducer/repressor comprises a tetR protein.

Viral Particle Production Methods

As described herein, in embodiments, the present disclosure relates to methods for making a viral particle packaging cell line.

As described herein, in embodiments, the present disclosure relates to methods for making a viral particle packaging and producer cell line. In embodiments, the cell is a mammalian cell. In embodiments, the cell is selected from a human embryonic kidney (HEK293), Chinese hamster ovary (CHO) E1A, E1 B+ engineered CHO-K1, Spodoptera fmgiperda (Sf9) cell line, baby hamster kidney (BHK), and Vero cell.

In embodiments, the E1A, E1 B+ cell is, without limitation, HEK293, E1A, E1 B+ engineered CHO-K1 , or Sf9 cell line. In embodiments, the HEK293 cell stably expresses tetR.

In embodiments, the cell is a Vero cell which stably expresses tetR. In embodiments, the cell is a Vero cell which stably expresses tetR and HSV-1 ICPO protein. In embodiments, the cell is a Vero cell as described in WO/2018/226638, which is incorporated by reference in its entirety.

In embodiments, the viral particle is an AAV of a suitable serotype. In embodiments, the AAV is selected from AAV1 , AAV2, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the AAV serotype is a natural serotype ora synthetically engineered serotype. In embodiments, the AAV is AAV2. In embodiments, the AAV is AAV9. In embodiments, the viral particle is an AAV of any of the AAV serotypes. Today, 12 AAV serotypes are known, and more than 100 variants have been identified. Different serotype capsids can infect different tissues or culture cells in different ways, which depend on the primary receptor and co-receptors on the cell surface or the intracellular trafficking pathway itself.

In embodiments, the viral particle is an AAV, optionally selected from AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9.

In embodiments, the first, second, and third nucleic acids are included in different expression vectors.

In embodiments, the first and second nucleic acids are included in a single expression vector, and the third nucleic acid is included in an expression vector that is different from the expression vector including the first and second nucleic acids. In embodiments, the expression vector is or comprises a plasmid or miniplasmid.

In embodiments, the first, second, and/or third nucleic acids are included as RNA. In embodiments, the first, second, and/or third nucleic acids are included as mRNA. In embodiments, the first, second, and/or third nucleic acids are included as modified mRNA (mmRNA). In embodiments, the mmRNA comprises one or more of a 5'-m7G cap (capO, cap1 , or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length.

In embodiments, the first, second, and/or third nucleic acids are included as DNA, optionally plasmid DNA or miniplasmid DNA. In embodiments, the first, second, and/or third nucleic acids are included as RNA, optionally mRNA, optionally mmRNA, optionally comprising one or more of a 5'-m7G cap (capO, cap1 , or cap2), a pseudouridine or n- methyl-pseudouridine substitution, and a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length and the first, second, and/or third nucleic acids are included as DNA, optionally plasmid DNA or miniplasmid DNA.

In embodiments, the first nucleic acid is included as RNA, optionally mRNA, optionally mmRNA and second and/or third nucleic acids are included as DNA, optionally plasmid DNA or miniplasmid DNA.

In embodiments, the transgene is flanked by AAV inverted terminal repeats (ITRs). In embodiments, the transgene encodes a complete polypeptide. In embodiments, the transgene is defective or substantially absent in a disease state. In embodiments, the present methods result in a cell that is capable of producing the transgene, e.g., for use in gene therapy, e.g., gene replacement therapy. In embodiments, there are provided methods of treatment of a genetic disease with a cell described herein.

In embodiments, the transgene has a size of about 200,000 base pairs or less. In embodiments, the transgene has a size of at least 200,000 base pairs. In embodiments, the transgene has a size of about 200,000 base pairs. In embodiments, the transgene has a size of about 150,000 base pairs or less. In embodiments, the transgene has a size of at least 150,000 base pairs. In embodiments, the transgene has a size of about 150,000 base pairs. In embodiments, the transgene has a size of about 100,000 base pairs or less. In embodiments, the transgene has a size of at least 100,000 base pairs. In embodiments, the transgene has a size of about 100,000 base pairs. In embodiments, the transgene has a size of about 75,000 base pairs or less. In embodiments, the transgene has a size of at least 75,000 base pairs. In embodiments, the transgene has a size of about 75,000 base pairs. In embodiments, the transgene has a size of about 50,000 base pairs or less. In embodiments, the transgene has a size of at least 50,000 base pairs. In embodiments, the transgene has a size of about 50,000 base pairs.

In embodiments, the present methods further comprising culturing the transfected cell in a medium that expands a population of the transfected cells to create a stably transfected packaging cell line or packaging and producer cell line.

In embodiments, the transfection comprises electroporation, nucleofection, lipofection, or calcium phosphate transfection.

In embodiments, the present methods are helper virus-free. Enzyme Capable of Performing Targeted Genomic Integration

In embodiments, the enzyme capable of performing targeted genomic integration is a mobile element enzyme. In embodiments, the mobile element enzyme has the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least about 80%, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, the mobile element enzyme comprises an amino acid other than serine at the position corresponding to position 2 of SEQ ID NO: 7. In embodiments, the amino acid is a non-polar aliphatic amino acid, optionally a non-polar aliphatic amino acid optionally selected from G, A, V, L, I and P, optionally A. In embodiments, the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 7. In embodiments, the enzyme has one or more mutations which confer hyperactivity. In embodiments, the enzyme has one or more amino acid substitutions selected from S8X1, C13X2, and N125Xs, or positions corresponding thereto relative to SEQ ID NO: 7. In embodiments, the enzyme has S8X1 substitution, or at a position corresponding thereto relative to SEQ ID NO: 7. In embodiments, the enzyme has C13X2 substitution, or at a position corresponding thereto relative to SEQ ID NO: 7. In embodiments, the enzyme has N125X₃ substitution, or at a position corresponding thereto relative to SEQ ID NO: 7. In embodiments, X₁ is selected from G, A, V, L, I, and P. In embodiments, X2 is selected from K, R, and H. In embodiments, X₃ is selected K, R, and H. In embodiments, Xi is P and X2 is R.

In embodiments, the enzyme is capable of inserting a donor DNA at a TA dinucleotide site.

In embodiments, the enzyme is capable of inserting a donor DNA at a TTAA tetranucleotide site. In embodiments, the enzyme comprises: (a) a targeting element, and (b) an enzyme that is capable of inserting the donor DNA (e.g., the donor DNA is a mobile element) comprising a gene, optionally at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS).

In embodiments, the targeting element targets the enzyme to a locus of interest.

In embodiments, the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, a transcription activator-like effector (TALE), or a Zinc finger.

In embodiments, the targeting element comprises a transcription activator-like effector (TALE) DNA binding domain (DBD). In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG.

In embodiments, the targeting element comprises CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) associated protein 9 (Cas9), or a variant thereof. A CRISPR/Cas9 tool only requires Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity. See Jinek et al. (2012) Science 337, 816-821 ; Chylinski etal. (2014) Nucleic Acids Res 42, 6091-6105. The inactivated form of Cas9, which is a nuclease-deficient (or inactive, or “catalytically dead” Cas9, is typically denoted as “dCas9,” has no substantial nuclease activity. Qi, L. S. et al. (2013). Cell 152, 1173-1183. CRISPR/dCas9 binds precisely to specific genomic sequences through targeting of guide RNA (gRNA) sequences. See Dominguez et al., Nat Rev Mol Cell Biol. 2016;17:5-15; Wang et al., Annu Rev Biochem. 2016;85:227-64. dCas9 is utilized to edit gene expression when applied to the transcription binding site of a desired site and/or locus in a genome. When the dCas9 protein is coupled to guide RNA (gRNA) to create dCas9 guide RNA complex, dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. Thus, dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription.

In embodiments, the targeting element comprises a nuclease-deficient Cas enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient (or inactive, or “catalytically dead” Cas, e.g., Cas9, typically denoted as “dCas” or “dCas9” ) guide RNA complex.

In embodiments, the targeting element comprises a Cas9 enzyme guide RNA complex. In embodiments, the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex.

Embodiments of the present disclosure make use of the ability of Zinc fingers or TALEs or Cas or dCas9/gRNA DBDs to target specific sites in a host genome. The DNA targeting ability of Zinc finger or TALE or Cas DBD or dCas9/gRNA DBD is provided by TALE repeat sequences (e.g., modular arrays) or gRNA which are linked together to recognize flanking DNA sequences. Each Zinc finger or TALE or gRNA can recognize certain base pair(s) or residue(s).

In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.

In embodiments, the enzyme and the targeting element are connected. In embodiments, the enzyme and the targeting element are fused to one another or linked via a linker to one another. In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)_n, where n is from about 1 to about 12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues.

In embodiments, at least one of the first nucleic acid and the second nucleic acid is in the form of a lipid nanoparticle (LNP). In embodiments, the first nucleic acid encoding the enzyme and the second nucleic acid encoding the donor DNA are in the form of the same LNP, optionally in a co-formulation.

Kits

In embodiments, the recombinant tetracycline inducer/repressor system described herein, a vector comprising the recombinant tetracycline inducer/repressor system described herein, cells produced in accordance with the method described herein, and/or components for generating cells with the method described herein, is included in a container, kit, pack, or dispenser together with instructions for administration.

Also provided herein are kits comprising: one or more genetic constructs encoding the present enzyme and donor DNA and instructions and/or reagents for the use of the same.

Also provided herein are kits comprising: i) a transfected cell in accordance with embodiments of the present disclosure, ii) instructions for the use of the transfected cell.

Furthermore, in embodiments, a kit is provided for creating a viral element, e.g., AAV, packaging cell line or a viral element, e.g., AAV, library of packaging cell lines, and instructions for creating a library. A subsequent user-defined transfection of the viral genome can be performed to generate a producer cell line. In embodiments, the kit includes instructions for transfection of a viral element, e.g., AAV, packaging cell line with a desired transgene.

In embodiments, a kit includes a viral element, e.g., AAV, library of packaging cell lines and instructions for creating a library.

In some aspects, a kit is provided that comprises an enzyme (e.g., without limitation, a recombinant mammalian mobile element enzyme) or a nucleic acid in accordance with embodiments of the present disclosure, and instructions for introducing DNA and/or RNA into a cell using the enzyme.

Definitions

The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs.

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.

As used herein, the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and/or additions at certain positions. The variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.

As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features. Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”

As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and/or compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.

SELECTED SEQUENCES

In embodiments, the present disclosure provides for any of the sequence provided herein, including the below, and a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

SEQ ID NO: 1. Nucleotide sequence of enhanced tetO promoter

The tet-operator-containing P5 promoter sequence (italics, nucleotides 146-300, SEQ ID NO: 9 below) with YY1 +1 element (underlined bold, nucleotides 291-300, CTCCATTTT) and modified TATA box element (underlined italics, nucleotides 211-217, TAT AT AA), insertion of enhancer element of HSV-1 ICP4 promoter (bold, nucleotides 9-145, SEQ ID NO: 8 below). The sequence is flanked by 5’-Pacl site (ttaattaa) and a 3’- Notl site (gcggccgc).

308 bp DNA

ORIGIN

1 TTAATTAAGC GGAACGGAAG CGGAAACCGC CGGATCGGGC GGTAATGAGA TGCCATGCGG

61 GGCGGGGCGC GGACCCACCC GCCCTCGCGC CCCGCCCATG GCAGATGGCG CGGATGGGCG

121 GGGCCGGGGG TTCGACCAAC GGGCCGGTCC TGTATTAGAG GTCACGTGAG TGTTTTGCGA

181 CATTTTGCGA CACCATGTGG TCACGCTGGG TATATAAGCA ACCGGTCTCC CTATCAGTGA

241 TAGAGATCTC CCTATCAGTG ATAGAGATCG GCCCGAGTGA GCACGCAGGG TCTCCATTTT

301 GCGGCCGC

SEQ ID NO: 9

SEQ ID NO: 2. Nucleotide sequence enhanced tetO promoter-1

The enhanced tet-operator-containing P5 promoter sequence (italics, nucleotides 150-299, SEQ ID NO: 10 below) with insertion of proximal cis-acting elements of the HCMV IE promoter (bold, nucleotides 9-149, SEQ ID NO: 11 below) at the 5’ end of P5 promoter with the modified TATA element (underlined italics, nucleotides 215-221) without YY1 +1 element. The sequence is flanked by 5’-Pacl site (ttaattaa) and a 3’-Notl site (gcggccgc).

307 bp DNA

ORIGIN

1 TTAATTAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT 61 GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC 121 AACTCCGCCC CATTGACGCA AATGGGCGGG GTCCTGTATT AGAGGTCACG TGAGTGTTTT 181 GCGACATTTT GCGACACCAT GTGGTCACGC TGGGTATATA AGCAACCGGT CTCCCTATCA 241 GTGATAGAGA TCTCCCTATC AGTGATAGAG ATCGGCCCGA GTGAGCACGC AGGGTCTCCG 301 CGGCCGC

SEQ ID NO: 10

SEQ ID NO: 11

SEQ ID NO: 3. Nucleotide sequence enhanced tetO promoter-2

325 bp DNA

ORIGIN

181 aggtcacgtg agtgt t ttgc gaca t t t tgc gacacca tgt ggtcacgctg ggta ta taaG 241 CAACCGGTCT CCCTATCAGT GATAGAGATC TCCCTATCAG TGATAGAGAT CGgcccgagt 301 gagcacgcag ggtctccGCG GCCGC

SEQ ID NO: 13 CC CCATTGACGT CAATGGGCGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGG

SEQ ID NO: 4. Nucleotide sequence enhanced tetO promoter-3.

ORIGIN

1 TTAATTAAGC GGAACGGAAG CGGAAACCGC CGGATCGGGC GGTAATGAGA TGCCATGCGG 61 GGCGGGGCGC GGACCCACCC GCCCTCGCGC CCCGCCCATG GCAGATGGCG CGGATGGGCG 121 GGGCCGGGGG TTCGACCAAC GGGCCggtcc tgta t tagag gtcacgtgag tgttttgcga. 181 ca.ttttgcga. cattttgcga ca t tt tgcga cacca tgtgg tcacgctggg ta ta taaGCA 241 ACCGGTCTCC CTATCAGTGA TAGAGATCTC CCTATCAGTG ATAGAGATCG gcccgagtga 301 gcacgcaggg tctccGCGGC CGC

SEQ ID NO: 14 ggtcc tgta t tagag gtcacgtgag cacca tgtgg tcacgctggg ta ta taa

SEQ ID NO: 8

SEQ ID NO: 5. Nucleotide sequence of tetO SV40 early major promoter LOCUS Modified SV40/TO promoter. The normal TAATTTTTTT (SEO ID NO: 17) element immediate upstream of the SV40 TATA box (underlined, nucleotides 252-258, tatataa) is replaced with aggcgccggg

(SEQ ID NO: 16) element (bold, nucleotides 242-251).

340 bp DNA

ORIGIN

SEQ ID NO: 6. Nucleotide sequence of modified HSV-2 ICP4 promoter

LOCUS HSV-2 1 CP4 promoter 366 bp DNA

ORIGIN

SEQ ID NO: 7: Myotis lucifugus mobile element enzyme protein (572 amino acids, positions 2, 8, 13, and 125, described in the text, are shown in bold and underline)

This invention is further illustrated by the following non-limiting examples.

EXAMPLES

Hereinafter, the present disclosure will be described in further detail with reference to examples. These examples are illustrative purposes only and are not to be construed to limit the scope of the present invention. In addition, various modifications and variations can be made without departing from the technical scope of the present invention.

Example 1: Gene Expression by the Present Tetracycline Inducer/Repressor System

Tetracycline repressor, tetR, expressing HEK293 cells, 293R-22 cells, were transiently transfected with a plasmid encoding eGFP under the control of the enhanced tetO-containing hCMV IE promoter. The 293R-22 cells were transfected with 0.1 pg of pCDNA4TO-EGFP and 1 .4 pg of pMP4 in eitherthe absence or presence of doxycycline.

FIG. 3 shows photos were taken at 48 h post-transfection. The results demonstrate that EGFP expression from the tetO-containing hCMV IE promoter is tightly suppressed in the absence of doxycycline, while high levels of eGFP expression are detected in the presence of doxycycline.

Example 2: Production of AAV Packaging and Producer Cell Lines Using Gene Editing Methods of the Present Disclosure

This example describes development of an AAV packaging cell line by utilizing AAV production methodologies in combination with the gene editing methods of the present disclosure.

AAV packaging and producer cell lines are manufactured by using HEK293 cell line transiently expressed with a three-plasmid system (“triple transfection”). The three plasmids encode the various components needed to produce AAV, including a genome/transgene vector, a packaging plasmid providing the REP and CAP genes, and a helper plasmid that supplies the adenovirus type 5 genes E2a, E4, and VA. Two other adenovirus genes required for AAV replication, E1 a and E1 b, are already expressed by HEK293. One technical challenge in developing packaging cell lines is preventing cell death due to the cytotoxicity of the E4, E2a, and REP genes. Therefore, cytotoxic genes are placed under the control of inducible promoter(s).

HEK293R-22 and V0R-124 cell lines (FIGs. 4A-4C) were used to engineer packaging cell lines. One of these packaging cell lines was used to generate producer cell lines by engineering cell lines with genome vectors.

FIG. 4A depicts the transient expression of EGFP under the control of the enhanced tetO promoter (P5TO) in Vero and V0R-124 cells. VOR-124 cells and CCL-81 Vero cells were transfected with 0.25 pg of pP5TO- EGFP and 1.25 pg of pMP4. Cells were cultured in the presence of 0.5 pg/ml of doxycycline (Dox). The results show that levels of EGFP expression from the P5TO promoter were significantly higher in VOR-124 cells than in CCL-81 Vero cells. VOR-124 is a CCL-81 Vero cel Is-deri ved stable cell line. VOR-124 expresses tetracycline repressor, tetR, and the HSV-1 major regulatory protein ICPO. ICPO is a potent transactivator that can activate gene expression in a promoter-sequence independent manner. The result indicates that the activity of the P5TO promoter can be efficiently enhanced by ICPO, which is stably expressed in VOR-124 cells. FIG. 4B and FIG. 4C depicts the transient expression of EGFP under the control of five different tetO promoter (P5TO) in 293R-22 cells transfected with 0.5pg of pP5TO-EGFP and 1 pg of carrier plasmid DNA. The results further demonstrate inducible eGFP expression under regulatable AAV P5-modified promoters in 293R-22 Cells with or without doxycycline.

Testing of AAV producer plasmids

Vector Engineering

The AAV2 REP/CAP and Ad5 E2A-IRES-E40rf6 genes were placed under the control of inducible tetO promoters (FIG. 5A). Since HEK293 already expresses E1 a and E1 b, vectors for these two genes are not needed for this cell line. However, VOR-124 requires E1 a and E1 b genes and are placed under the control of a constitutive promoter. Insulators are included in the construct design (FIG. 5A) to, e.g., prevent the potential influence of cis elements near the genome's integration sites. A constitutive promoter-GFP reporter construct was created as a control to evaluate if the synthetic tetO promoter is leaky and evaluate the relative level of induction that can be achieved (FIG. 3, FIG. 4B and FIG. 4C). Results show that eGFP expression from tetO- promoter can be tightly suppressed in the absence of doxycycline, while high levels of eGFP expression are detected in the presence of doxycycline (FIGs. 4A-4C).

FIGs. 5A-5B depict plasmid vectors used for transfection. FIG. 5A shows a complete AAV capsid producer donor element. This 13.2 kb DNA element contains two ITRs and two insulators (HS4, D4Z4) that flank components that produce AAV. The AAV2 REP/CAP gene is driven by a tetracycline-inducible promoter (enhanced tetO), and the bicistronic adenovirus 5 E2A-IRES-E4 gene is driven by an inducible tetO SV40 promoter. FIG. 5B shows the 6.2 kb AAV2 CAG-GFP transgene flanked by AAV ITR.

FIG. 3 depicts 293R-22 cells were transiently transfected with a plasmid with eGFP under the control of the enhanced tetO-containing hCMV I E promoter. The 293R-22 cells were transfected with 0.1 pg of pCDNA4TO- EGFP and 1.4 pg of pMP4 in either the absence or presence of doxycycline (Dox). Photos were taken 48 h post-transfection. The results demonstrate that GFP expression from the tetO-containing hCMV IE promoter can be tightly suppressed in the absence of doxycycline. In contrast, high levels of EGFP expression are detected in the presence of doxycycline.

Results

The following procedures and methods were used to generate the data shown in FIGs. 6A-6D:

Day 1 : Seeded 5 x 60 mm dishes of passage 37 293R-22 cells at 1 .5 x 10e⁶ cells/dish using normal 293 cells growth medium.

Day 2: Changed media using 4 ml/dish antibiotics-free DMEM containing 10% FBS. Incubated at 37 °C. 2 hours later, removed the medium, washed once with 1.5 ml/dish of serum-free and antibiotic-free DMEM. Then, transfected 4 x 60 mm dishes with lipofectamine 2000 according to the laboratory protocol and the following:

1) Three (3) pg of complete AAV capsid producer donor element with inducible tetracycline promoters (VB211213-1121dbe) (FIG. 5A).

2) One and a half (1.5) pg of AAV2 ITR CMV-GFP transgene (VB211202-1457fww) (FIG. 5B).

3) 3 pg of pMF-tetR per dish.

Transfection was carried out at 37 °C for 3 hours, followed by removal of transfection medium and adding of 5 ml/dish of normal 10 x 3 growth medium in either the absence (Dox-) or presence (Dox+) of 0.5 pg/ml of doxycycline.

During the removal and addition of medium steps, some of transfected 293R-22 cells were detached from the plates.

Day 3: 60% to 70% of cells were eGFP-positive in Dox+ and Dox- dishes. There were no differences between Dox+ and Dox- dishes with respect to eGFP intensity. When checked at the transfected cells under the light microscope, it was observed that cells looked slightly more stressed in the Dox+ dishes compared to the Dox- dishes.

Cells were harvested and placed in a 15 ml centrifuge tube. Cells were designated AAV-eGFP/DOX+ and AAV-eGFP/DOX- respectively. Cells were stored at -80 °C.

Day 4-7: Stocks were frozen and thawed three times, followed by centrifugation at 2150 rpm for 12 min. Each infectious supernatant was transferred to a new 15 ml centrifuge tube and mixed well. The supernatant was transferred to 5 x 2 ml cryogenic vials and 1 x 5 ml round-bottom tube. Samples were stored on ice, then at -80 °C.

Day 4: 1). Untransfected U-2 OS cells were seeded on 60 mm dishes (n=8) at 6 x 10⁵ cells/dish with 10 x 3 medium, and 2) untransfected 293R-22 cells were seeded at 60 mm dishes (n=12, passage 8) at 5 x 10e⁵ cells/dish with 10 x 3 medium.

Day 7: Untransfected cells were visualized. There were 2.5 x 10⁶ U-2 OS cells per dish and about 2.5 x 10⁶ 293R-22 cells per dish. These cells were infected with the AAV-eGFP/DOX+ and AAV-eGFP/DOX- supernatant solutions.

1) 60 mm dishes of 293R-22 cells were transduced or infected with AAV-EGFP/DOX+ and AAV- eGFP/DOX- stocks at varying volumes of supernatant (0.5 ml/dish, 200 pl/dish, 100 pl/dish, 50 pl/dish, 20 pl/dish, and 10 pl/dish) in an inoculation volume of 0.6 ml/dish.

2) 60 mm dishes of U2OS cells were transduced or infected with AAV-eGFP/DOX+ and AAV- eGFP/DOX- stocks at varying volumes of supernatant (0.5 ml/dish, 200 pl/dish, 100 pl/dish, and 50 pl/dish in an inoculation volume of 0.6 ml/dish.

3) Cells were incubated for 1.5 hours at 37 °C. Inoculation medium was removed from each dish, followed by adding 4 ml/dish of fresh 10 x 3 medium.

Day 10-12: When checked under the green fluorescent microscope, 1 to 5 positive eGFP-positive cells were occasionally identified in the 293R-22 and U-2 OS dishes infected with 500 pl of AAV-eGFP/DOX+ stock. No eGFP-positive cells were detected in 293R-22 and U-2 OS dishes infected with 500 pl of AAV-eGFP/DOX- stock (FIGs. 6A-6D).

The presence of eGFP-positive 293R-22 cells and eGFP-positive U-2 OS cells after transduced with AAV- eGFP/DOX+ stock in FIGs. 6A-6D indicates that transfection of 293R-22 cells with the two plasmids described in FIG 5A and FIG 5B can lead to encapsulation of AAV ITR CAG-GFP by AAV. The production of eGFP-positive U-2 OS and 293R-22 cells after transduction using supernatant from AAV-eGFP/DOX+ stock but not AAV-eGFP/DOX- stock indicates that AAV production and encapsulation occurred after the initial transfection. This data further supports that the enhanced tetO and the tetO SV40 early major promoter induce the formation of infectious AAV particles. FIGs. 6A-6D depict GFP visualization hours after transduction of 293R-22 and U-2 OS cells with supernatant from cells transfected with complete AAV capsid producer donor element with inducible tetracycline promoters and AAV2 ITR CMV-GFP transgene. FIG. 6A and FIG. 6C show transduction with AAV- EGFP/DOX+ supernatant. FIG. 6B and FIG. 6D show transduction of AAV-EGFP/DOX- supernatant. FIG. 6A and FIG. 6B are transduced 293R-22 cells visualized at 72 hours. FIG. 6C and FIG. 6D are transduced U-2 OS cells visualized at 92 hours.

Production of AAV

Complete AAV capsid producer donor element with inducible tetracycline promoters (VB211213-1121 dbe) (FIG. 5A) will be integrated into HEK293 and VOR-124 cell lines. The producer cell lines will be tested for AAV production. The AAV2 ITR CMV-GFP transgene (VB211202-1457fww) plasmid (FIG. 5B) will be utilized. Experiments will be carried out to evaluate these vectors for transfection efficiency, viral productivity, and fine-tune culture conditions for scalability. Additional experiments will evaluate recovery and separation of empty/full capsids. Quality control (QC) parameters will be checked using various assays (e.g., without limitation, qPCR, ELISA, SPR, HPLC, and TEM).

EQUIVALENTS

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims. INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

What is claimed is:

1 . A composition comprising a recombinant tetracycline inducer/repressor system comprising:

(i) at least one of a P5 promoter or SV40 early promoter, or a functional variant or fragment thereof,

(ii) a modified TATA box element, and

(iii) at least one tet operator, wherein the tetracycline inducer/repressor system provides substantially non-leaky control of expression of a gene in the vector.

2. A method of making a viral particle packaging cell line, the method comprising transfecting an E1A+, E1 B+ cell with:

(a) a first nucleic acid encoding an enzyme capable of performing targeted genomic integration, and

(b) a second nucleic acid encoding a donor DNA under control of a recombinant tetracycline inducer/repressor system and comprising terminal ends recognized by the enzyme, wherein the donor DNA comprises:

(i) inducible viral replication (REP) and Capsid (CAP) genes,

(ii) one or more adenoviral auxiliary genes, optionally selected from E4, E2A, and VA of an AAV, and

(iii) one or more insulators, optionally selected from HS4, D4Z4, and wherein the tetracycline inducer/repressor system comprises:

(ii) a modified TATA box element, and

(iii) at least one tet operator, to thereby result in a viral particle packaging cell line.

3. The method of claim 2, the method further comprising converting the packaging cell line into a producer cell by contacting the packaging cell line with a third nucleic acid encoding a donor DNA comprising a transgene flanked by AAV inverted terminal repeats (ITRs) to result in a transfected cell that expresses the transgene in association with the viral particle.

4. The method of claim 2 or 3, wherein the second nucleic acid is substantially of the form as show in FIG. 2, or a functional equivalent thereof.

5. A method of making a viral particle packaging and producer cell line, the method comprising transfecting an E1A, E1 B+ cell with:

(b) a second nucleic acid encoding a donor DNA under control of a recombinant tetracycline inducer/repressor system and comprising terminal ends recognized by the enzyme and a transgene disposed between the terminal end, wherein the donor DNA comprises:

(i) inducible viral replication (REP) and Capsid (CAP) genes,

(ii) a modified TATA box element, and

(iii) at least one tet operator, to thereby result in a transfected cell that expresses the transgene in association with a viral particle.

6. The method of any one of claims 2-5, wherein the tetracycline inducer/repressor provides substantially non-leaky control of expression. The composition of claim 1 or the method of any one of claims 2-6, wherein the tetracycline inducer/repressor is induced by doxycycline. The composition of claim 1 or 7 or the method of any one of claims 2-7, wherein the tetracycline inducer/repressor provides substantially non-leaky control of expression of one or more of components of the donor DNA. The composition of claim 1 or 7-8 or the method of any one of claims 2-8, wherein the tetracycline inducer/repressor provides substantially non-leaky control of expression of one or more of REP, CAP, E1A, E1 B, E4, E2A, and VA. The composition of claim 1 or 7-9 or the method of claim 9, wherein the tetracycline inducer/repressor provides substantially non-leaky control of expression of one or more of E4, E2A, and REP. The composition of claim 1 or 7-10 or the method of any one of the above claims, wherein the leakiness is assessed by measuring a transcription level at the promoter inactive state. The composition of claim 1 or 7-11 or the method of any one of the above claims, wherein the leakiness is assessed by measuring a transcription level at the promoter active state and inactive state and comparing the same. The composition of claim 1 or 7-12 or the method of any one of the above claims, wherein the leakiness is assessed by measuring a transcription level in the absence of doxycycline. The composition of claim 1 or 7-13 or the method of any one of the above claims, wherein the leakiness is assessed by measuring a transcription level in the presence of doxycycline and the absence of doxycycline and comparing the same. The composition of claim 1 or 7-14 or the method of any one of the above claims, wherein the ratio of expression in the promoter inactive state to expression in the promoter active state to is at least about 1 : 100, or at least about 1 : 500, or at least about 1 : 1000, or at least about 1 : 5000, or at least about 1 : 10000. The composition of claim 1 or 7-15 or the method of any one of the above claims, wherein the ratio of expression in the doxycycline absent state to expression in the doxycycline present state is at least about 1 : 100, or at least about 1 : 500, or at least about 1 : 1000, or at least about 1 : 5000, or at least about 1 : 10000. The composition of claim 1 or 7-16 or the method of any one of the above claims, wherein the expression is less leaky than that observed in an equivalent tetracycline inducer/repressor system having a strong promoter, instead of P5 promoter or SV40 early promoter. The composition of claim 1 or 7-17 or the method of any one of the above claims, wherein the strong promoter is the human cytomegalovirus (CMV) immediate-early promoter. The composition of claim 1 or 7-18 or the method of any one of the above claims, wherein the expression is less leaky than that observed in the construct of U.S. Patent No. 5,972,650. The composition of claim 1 or 7-19 orthe method of any one ofthe above claims, wherein the tetracycline inducer/repressor comprises a tet operator sequence positioned at least about 4 to about 12 nucleotides, or at least about 4 to about 6 nucleotides 3' to the beginning of the modified TATA box element, optionally about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 nucleotides 3' to the beginning of the modified TATA box element. The composition of claim 1 or 7-20 orthe method of any one ofthe above claims, wherein the tetracycline inducer/repressor further comprises a gene 3' to the tet operator sequence, the gene optionally being one or more of E4, E2A, and REP. The composition of claim 1 or 7-21 or the method of any one of the above claims, wherein the at least one of a P5 promoter or SV40 early promoter is 5' of the modified TATA box element or wherein the at least one of a P5 promoter or SV40 early promoter contains a modified TATA box. The composition of claim 1 or 7-22 or the method of any one of claims 20-22, wherein: the at least one of a P5 promoter or SV40 early promoter is 5' of the modified TATA box element or wherein the at least one of a P5 promoter or SV40 early promoter contains a modified TATA box; the modified TATA box element is 5' of the one or more tet operator sequences; and/or the one or more tet operator sequences is 5' of a gene, optionally one or more components of the donor DNA, or one or more of one or more of REP, CAP, E1A, E1 B, E4, E2A, and VA, or one or more of E4, E2A, and REP. The composition of claim 1 or 7-23 or the method of any one of claims 1 -23, wherein the modified TATA box element has the nucleotide sequence of TATATAA or TATATAG.

25. The composition of claim 1 or 7-24 or the method of claim 24, wherein the modified TATA box element has the nucleotide sequence of aggcgccggg (SEQ ID NO: 16), or a functional variant thereof, disposed 5' therefrom.

26. The composition of claim 1 or 7-25 or the method of any one of claims 2-25, wherein the modified TATA box element does not have the nucleotide sequence of TATTTAA orTATTTAT.

27. The composition of claim 1 or 7-26 or the method of any one of claims 2-25, wherein the modified TATA box element does not have the nucleotide sequence of TAATTTTTTT (SEQ ID NO: 17).

28. The composition of claim 1 or 7-27 or the method of any one of claims 2-27, wherein the promoter in tetracycline inducer/repressor system further comprises one or more enhancer elements.

29. The composition of claim 1 or 7-28 or the method of claim 28, wherein the enhancer element comprises one or more cis-acting elements, optionally selected from an Sp1 binding site, GC rich sequence, GCGGAAC motif, TAATGARAT element, AP1 binding site, and CCAAT box element.

30. The composition of claim 1 or 7-29 or the method of claim 29, wherein the enhancer element comprises about 1 to about 5 Sp1 binding sites, optionally about 1 , or about 2, or about 3, or about 4, or about 5 Sp1 binding sites.

31. The composition of claim 1 or 7-30 or the method of claim 29 or 30, wherein the enhancer element comprises about 1 or about 2 GC rich sequences.

32. The composition of claim 1 or 7-31 or the method of any one of claims 28-31 , wherein the enhancer elements are derived from an HSV-1 ICP4 promoter.

33. The composition of claim 1 or 7-32 or the method of any one of claims 2-32, wherein the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 1, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto; the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 2, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto; the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 3, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto; the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 4, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto; or the tetracycline inducer/repressor comprises the nucleotide sequence of SEQ ID NO: 5, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto. The composition or method of any one of claims 1-33, wherein the tetracycline inducer/repressor comprises one or more genes encoding tet repressor (tetR), or a codon-optimized tetR. A cell comprising the tetracycline inducer/repressor of any one of claims 1-34. The method of any one of claims 2-34, wherein the cell is selected from a human embryonic kidney (HEK293), Chinese hamster ovary (CHO) E1A, E1 B+ engineered CHO-K1, Spodoptera fmgiperda (Sf9) cell line, baby hamster kidney (BHK), and Vero cell, optionally wherein, the cell stably expresses tetR. The method of any one of claims 2-34 or 36, wherein the viral particle is an AAV, optionally selected from AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9. The method of any one of claims 2-34 or 36-37, wherein the first, second, and third nucleic acids are included in different expression vectors. The method of any one of claims 2-34 or 36-38, wherein the first and second nucleic acids are included in a single expression vector, and the third nucleic acid is included in an expression vector that is different from the expression vector including the first and second nucleic acids. The method of claim 39, wherein the expression vector is or comprises a plasmid or miniplasmid.

41. The method of any one of claims 39 or 40, wherein the first, second, and/or third nucleic acids are included as RNA, optionally mRNA, optionally modified mRNA (mmRNA), optionally comprising one or more of a 5'-m7G cap (capO, cap1 , or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length.

42. The method of any one of claims 39-41 , wherein the first, second, and/or third nucleic acids are included as DNA, optionally plasmid DNA or miniplasmid DNA.

43. The method of any one of claims 39-42, wherein the first, second, and/or third nucleic acids are included as RNA, optionally mRNA, optionally mmRNA, optionally comprising one or more of a 5'-m7G cap (capO, cap1 , or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length and the first, second, and/or third nucleic acids are included as DNA, optionally plasmid DNA or miniplasmid DNA.

44. The method of any one of claims 39-43, wherein the first nucleic acid is included as RNA, optionally mRNA, optionally mmRNA and second and/or third nucleic acids are included as DNA, optionally plasmid DNA or miniplasmid DNA.

45. The method of any one of claims 39-44, wherein the transgene is flanked by AAV inverted terminal repeats (ITRs).

46. The method of any one of claims 39-45, wherein the transgene encodes a complete polypeptide.

47. The method of any one of claims 39-46, wherein the transgene is defective or substantially absent in a disease state.

48. The method of any one of claims 2-34 or 36-47, further comprising culturing the transfected cell in a medium that expands a population of the transfected cells to create a stably transfected packaging cell line or packaging and producer cell line.

49. The method of any one of claims 2-34 or 36-48, wherein the transfection comprises electroporation, nucleofection, lipofection, or calcium phosphate transfection.

50. The method of any one of claims 2-34 or 36-49, wherein the method is helper virus-free.

51. The method of any one of claims 2-34 or 36-50, wherein the enzyme capable of performing targeted genomic integration is a mobile element enzyme.

52. The method of any one of claims 2-34 or 36-51 , wherein the mobile element enzyme has the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least about 80%, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

53. The method of claim 52, wherein the mobile element enzyme comprises an amino acid other than serine at the position corresponding to position 2 of SEQ ID NO: 7.

54. The method of claim 53, wherein the amino acid is a non-polar aliphatic amino acid, optionally a nonpolar aliphatic amino acid optionally selected from G, A, V, L, I and P, optionally A.

55. The method of claim 53 or 54, wherein the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 7.

56. The method of any one of claims 52-55, wherein the enzyme has one or more mutations which confer hyperactivity.

57. The method of any one of claims 52-56, wherein the enzyme has one or more amino acid substitutions selected from S8X1, C13X2, and N125X3, or positions corresponding thereto relative to SEQ ID NO: 7.

58. The method of claim 57, wherein the enzyme has S8X1 substitution, or at a position corresponding thereto relative to SEQ ID NO: 7.

59. The method of claim 57, wherein the enzyme has C13X2 substitution, or at a position corresponding thereto relative to SEQ ID NO: 7.

60. The method of claim 57, wherein the enzyme has N125X3 substitution, or at a position corresponding thereto relative to SEQ ID NO: 7.

61. The method of claim 58, wherein Xi is selected from G, A, V, L, I, and P.

62. The method of claim 59, wherein X2 is selected from K, R, and H.

63. The method of claim 60, wherein X3 is selected K, R, and H.

64. The method of claim 57, wherein: Xi is P and X2 is R.

65. The method of any one of claims 52-64, wherein the enzyme is capable of inserting a donor DNA at a TA dinucleotide site.

66. The method of any one of claims 52-64, wherein the enzyme is capable of inserting a donor DNA at a TTAA tetranucleotide site.

67. The method of any one of claims 52-66, wherein the enzyme comprises:

(a) a targeting element, and

(b) an enzyme that is capable of inserting the donor DNA, optionally a mobile element, comprising a gene, optionally at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS).

68. The method of claim 67, wherein the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, a transcription activator-like effector (TALE), or a zinc finger.

69. The method of claim 68, wherein the targeting element comprises a transcription activator-like effector (TALE) DNA binding domain (DBD).

70. The method of claim 68, wherein the targeting element comprises a Cas9 enzyme guide RNA complex.

71 . The method of claim 70, wherein the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex.

72. The method of any one of claims 67-71 , wherein the GSHS is in an open chromatin location in a chromosome.

73. The method of claim 72, wherein the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.

74. The method of any one of claims 67-73, wherein the enzyme and the targeting element are connected.

75. The method of any one of claims 67-74, wherein the enzyme and the targeting element are fused to one another or linked via a linker to one another.

76. The method of claim 75, wherein the linker is a flexible linker.

77. The method of claim 76, wherein the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)_n, where n is from about 1 to about 12.

78. The method of claim 77, wherein the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues.

79. The method of any one of claims 2-34 or 36-78, wherein at least one of the first nucleic acid and the second nucleic acid is in the form of a lipid nanoparticle (LNP).

80. The method of any one of claims 2-34 or 36-79, wherein the first nucleic acid encoding the enzyme and the second nucleic acid encoding the donor DNA are in the form of the same LNP, optionally in a coformulation.

81. A composition comprising the nucleotide sequence of SEQ ID NO: 1 , or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

82. A composition comprising the nucleotide sequence of SEQ ID NO: 2, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

83. A composition comprising the nucleotide sequence of SEQ ID NO: 3, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

84. A composition comprising the nucleotide sequence of SEQ ID NO: 4, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

85. A composition comprising the nucleotide sequence of SEQ ID NO: 5, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

86. A composition comprising the nucleotide sequence of SEQ ID NO: 6, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

87. A composition comprising the nucleotide sequence of SEQ ID NO: 8, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

88. A composition comprising the nucleotide sequence of SEQ ID NO: 9, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

89. A composition comprising the nucleotide sequence of SEQ ID NO: 10, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

90. A composition comprising the nucleotide sequence of SEQ ID NO: 11 , or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

91 . A composition comprising the nucleotide sequence of SEQ ID NO: 12, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. A composition comprising the nucleotide sequence of SEQ ID NO: 13, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. A composition comprising the nucleotide sequence of SEQ ID NO: 14, or a functional variant or fragment thereof, or sequence having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% identity thereto, or at least about 98% identity thereto, or at least about 99% identity thereto, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.