Methods for regulating production of adeno-associated virus

Abstract

FIELD: biotechnology.SUBSTANCE: method for regulating the production of recombinant adeno-associated virus (rAAV) vector particles is described, wherein the method comprises: introducing into a cell: rAAV containing a gene of interest; and a nucleic acid encoding a fusion protein, wherein the fusion protein contains an AAV protein and a degradation domain dependent on a degradation ligand, culturing the cell under conditions suitable for the production of rAAV vector particles; and bringing the cell into contact with a degradation ligand, wherein the degradation ligand binds to the degradation domain, regulating the expression of the AAV protein and thereby regulating the production of rAAV vector particles. A cell producing rAAV is also described, wherein the cell comprises a nucleic acid encoding a fusion protein comprising an AAV protein and a degradation ligand-dependent degradation domain.EFFECT: regulation of Rep protein accumulation to avoid Rep-mediated cytotoxicity, resulting in regulated rAAV production.65 cl, 20 dwg, 7 ex

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority over

[0003] U.S. Provisional Application No. 63 / 350,849, filed June 9, 2022, and U.S. Provisional Application No. 63 / 209,735, filed June 11, 2021, both of which are incorporated herein by reference in their entireties and to which priority is claimed.

[0004] 1. FIELDS OF THE INVENTION

[0005] The currently disclosed subject matter relates to compositions and methods for regulating the production of recombinant adeno-associated virus (rAAV) in cell culture. In particular, the currently disclosed subject matter relates to strategies for overcoming AAV Rep protein-mediated cytotoxicity by reversibly post-translationally regulating AAV Rep protein expression, resulting in regulated rAAV production.

[0006] 2. LEVEL OF TECHNOLOGY

[0007] There are numerous AAV production systems used to produce rAAV in cell culture. These include transient plasmid transfection into human embryonic kidney (HEK) 293 cells, Hela cell lines, BHK21-based platforms, and baculovirus-based production systems. Each of these systems has strengths and weaknesses. For example, given the importance of the adenovirus E1a protein in initiating rAAV production, E1a-expressing cells, such as HEK293 cells, are attractive for rAAV production because they eliminate the need to otherwise introduce the E1a gene into the host cell genome. E1a-expressing cells, such as HEK293 cells, may also offer ease of growth and adaptation to growth in suspension. However, efforts to generate stable and passage-maintained rAAV-producing cell lines expressing E1a have been hampered by cellular toxicity caused by E1a-induced accumulation of AAV Rep protein.In view of the above, there is a need in the art for new rAAV production strategies that can regulate the accumulation of Rep protein to avoid Rep-mediated cytotoxicity, resulting in regulated rAAV production.

[0008] 3. ESSENCE OF THE INVENTION

[0009] In some embodiments, the present invention relates to methods for regulating the production of recombinant adeno-associated virus (rAAV) vector particles, the method comprising: introducing into a cell: rAAV containing a gene of interest and a nucleic acid encoding a fusion protein, wherein the fusion protein comprises an AAV protein and a degradation domain dependent on a degradation ligand, culturing the cell under conditions suitable for the production of rAAV vector particles; and contacting the cell with a degradation ligand, wherein the degradation ligand binds to the degradation domain, regulating the expression of the AAV protein and thereby regulating the production of rAAV vector particles.

[0010] In some embodiments, a nucleic acid encoding a fusion protein comprises a Rep protein, a linker, and a degradation ligand-dependent degradation domain. In some embodiments, the degradation ligand-dependent degradation domain is derived from FKBP. In some embodiments, the degradation ligand-dependent degradation domain is DHFR. In some embodiments, the degradation ligand-dependent degradation domain is an auxin-inducible degradation domain. In some embodiments, the degradation ligand is a small molecule ligand. In some embodiments, the small molecule is Shield1. In some embodiments, the small molecule is TMP. In some embodiments, the small molecule is auxin. In some embodiments, the small molecule is dTag13.

[0011] In some embodiments, a nucleic acid encoding a fusion protein comprises a Cap protein, a linker, and a degradation ligand-dependent degradation domain. In some embodiments, the degradation ligand-dependent degradation domain is derived from FKBP. In some embodiments, the degradation ligand-dependent degradation domain is DHFR. In some embodiments, the degradation ligand-dependent degradation domain is an auxin-inducible degradation domain. In some embodiments, the degradation ligand is a small molecule ligand. In some embodiments, the small molecule is Shield1. In some embodiments, the small molecule is TMP. In some embodiments, the small molecule is auxin. In some embodiments, the small molecule is dTag13.

[0012] In some embodiments, a nucleic acid encoding a fusion protein comprises a helper protein, a linker, and a degradation ligand-dependent degradation domain. In some embodiments, the helper protein is E2. In some embodiments, the degradation ligand-dependent degradation domain is derived from FKBP. The degradation ligand-dependent degradation domain is DHFR. In some embodiments, the degradation ligand-dependent degradation domain is an auxin-inducible degradation domain. In some embodiments, the degradation ligand is a small molecule ligand. In some embodiments, the small molecule is Shield1. In some embodiments, the small molecule is TMP. In some embodiments, the small molecule is auxin. In some embodiments, the small molecule is dTag33.

[0013] In some embodiments, the cell is a cell expressing E1a. In some embodiments, the cell expressing E1a is a HEK293 cell.

[0014] In some embodiments, the Rep protein is a Rep78, Rep68, Rep52, or Rep40 protein.

[0015] In some embodiments, the degradation ligand-dependent degradation domain is fused to the C-terminal end of the AAV protein. In some embodiments, the degradation ligand-dependent degradation domain is fused to the N-terminal end of the AAV protein.

[0016] In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker.

[0017] In some embodiments, the methods of the present invention comprise introducing a nucleic acid encoding a Cap protein into a cell. In some embodiments, the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using at least one plasmid. In some embodiments, the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using the same plasmid. In some embodiments, the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using separate plasmids. In some embodiments, the gene encoding the AAV fusion protein and / or the cap gene are under the control of a regulatory element. In some embodiments, the regulatory element is a promoter.In some embodiments, the regulatory element is a Tet response element.

[0018] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the mammalian cell is an HEK cell.

[0019] In some embodiments, the present invention relates to a cell that produces rAAV, wherein the cell comprises a nucleic acid encoding a fusion protein comprising an AAV protein and a degradation ligand-dependent degradation domain. In some embodiments, the nucleic acid encoding the fusion protein comprises an AAV protein, a linker, and a degradation ligand-dependent degradation domain. In some embodiments, the degradation ligand-dependent degradation domain is derived from FKBP. In some embodiments, the degradation ligand-dependent degradation domain is derived from FKBP. In some embodiments, the degradation ligand-dependent degradation domain is DHFR. In some embodiments, the degradation ligand-dependent degradation domain is an auxin-inducible degradation domain. In some embodiments, the degradation ligand is a small molecule ligand.In some embodiments, the small molecule is Shield1. In some embodiments, the small molecule is TMP. In some embodiments, the small molecule is auxin. In some embodiments, the small molecule is dTag13.

[0020] In some embodiments, the cell,

[0021] producing rAAV is a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK cell. In some embodiments, the cell is an E1a-expressing cell. In some embodiments, the E1a-expressing cell is a HEK293 cell.

[0022] In some embodiments, a cell producing rAAV comprises a ligand-dependent degradation domain fused via a linker to the C-terminal end of the AAV protein. In some embodiments, the ligand-dependent degradation domain is fused via a linker to the N-terminal end of the AAV protein. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker.

[0023] In some embodiments, the cell comprises a nucleic acid encoding the Cap protein. In some embodiments, the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using at least one plasmid. In some embodiments, the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using the same plasmid. In some embodiments, the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using separate plasmids. In some embodiments, the gene encoding the AAV fusion protein and / or the cap gene are under the control of a regulatory element. In some embodiments, the regulatory element is a promoter. In some embodiments, the regulatory element is a Tet response element.

[0024] 4. BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0025] Figure 1 shows examples of plasmid constructs that find use in the method and systems of the present invention.

[0026] Figure 2 depicts a schematic of the experimental flow to validate rAAV production using Degron constructs such as those shown in Figure 1.

[0027] Figure 3 shows that rAAV can be produced by transfecting mammalian cells carrying the Rep and Cap genes on separate plasmids.

[0028] Figure 4 shows that Rep-degron constructs can be used to produce AAV, and the accumulation of Rep protein can be regulated by the addition of the Shield-1 molecule.

[0029] Figure 5 shows the post-translational regulation of expression of Rep constructs mediated by Shield-1, wherein the construct encodes Rep with a C-terminal degron fusion or Rep with an N-terminal degron fusion.

[0030] Figure 6 shows the post-translational regulation of Rep protein expression mediated by Shield-1, with or without fusion of the C-terminal degron, and wherein Rep is present in the same construct as Cap or is present in a separate construct.

[0031] Figure 7 shows that rAAV production can be modulated by fusing the C-terminal degron to Rep and adding increasing concentrations of Shield-1; and that increasing the concentration of the Rep-degron plasmid, with Rep expression under the control of the CMV promoter, results in decreased rAAV production.

[0032] Figure 8 shows the production of rAAV using the Rep protein with or without fusion of the C-terminal degron and the inclusion of rigid or flexible linkers between Rep and the degron.

[0033] Figure 9 shows the production of rAAV using the Rep protein with or without fusion of the N-terminal degron and the inclusion of rigid or flexible linkers between Rep and the degron.

[0034] Figure 10 shows the regulation of C-terminal degron Rep constructs and N-terminal degron Rep constructs by Shield-1.

[0035] Figure 11 shows the possible size changes in Western blot after adding a degron tag to the N-terminus or C-terminus of the Rep protein. Adding a degron to the N-terminus of the Rep protein (N-degron) changes the molecular weight of the large Rep protein but not the small Rep protein. The molecular weight of the small Rep protein is not affected by the N-terminal tag because the p19 promoter, which drives the expression of the small Rep protein, is located inside the Rep gene. The small Rep protein does not have the same N-terminal sequence as the large Rep protein. In contrast, adding a degron to the C-terminus of the Rep protein (C-degron) changes the molecular weight of both small and large Rep proteins because the C-terminus is the same for both. Addition of small molecules such as Shieldl or TMP to the cell culture medium can inhibit protein degradation, and the band intensity will change accordingly. The example shown in this paper is a degron obtained from FKBP.

[0036] Figures 12A-12D depict the regulation of AAV production by Rep proteins using an FKBP-derived degron. Figure 12A depicts a Western blot of N-terminal Rep proteins tagged with the FKBP degron. Figure 12B depicts a Western blot of C-terminal Rep proteins tagged with the FKBP degron. Figure 12C shows the AAV titers of the samples in Figures 12A and 12B. Figure 12D shows the AAV titers with Rep and Cap plasmids transfected at different ratios.

[0037] Figures 13A-13B depict the regulation of AAV production by Rep proteins using a degron derived from E. coli DHFR (ecDHFR degron). Figure 13A depicts a Western blot of N-terminal Rep proteins tagged with the ecDHFR degron. Figure 13B depicts a Western blot of C-terminal Rep proteins tagged with the ecDHFR degron. Figure 13B shows the AAV titers of the samples in Figures 13A and 13B.

[0038] Figures 14A-14B depict the regulation of AAV production by Rep proteins using an auxin-based degron and an ecDHFR degron. Figure 14A depicts a Western blot of Rep labeled with an auxin-inducible degron or an ecDHFR degron. Figure 14B shows the AAV titers of the samples from Figure 14A.

[0039] Figure 15 shows the effects of different doses of Shieldl and dTag13 on AAV production. dTAG-13 is a small molecule that can target mutant FKBP sequences for ubiquitin-mediated degradation. It may function by linking the target protein sequence to the E3 ubiquitin ligase, cereblon. dTAG-13 can lead to the degradation of FKBP fusion proteins and proteins fused to it.

[0040] Figures 16A-16B show a Western blot of Rep with a C-terminal degron under the control of a promoter containing a Tet response element (TRE3G) exposed to Tet protein and a single concentration of doxycycline (Figure 16A), and the AAV titers of the samples of Figure 16A (Figure 16B).

[0041] Figures 17A-17B depict a Western blot of Rep with a C-terminal degron under the control of the TRE3G promoter and the effect of Tet protein over a range of doxycycline concentrations (Figure 17A) and AAV titers of the samples of Figure 17A (Figure 17B).

[0042] Figures 18A-18B show the effect of the p5 promoter on Rep protein levels in the Tet system, specifically the TRE3G-Rep-degron system (Figure 18A); Western blot of Rep constructs with a C-terminal degron and the TRE3G promoter (Figure 18B); and AAV titers of the samples of Figure 18B (Figure 18C).

[0043] Figures 19A-1EB. Figure 19A shows DBP protein expression observed in the E2A gene tagged with an FKBP-derived degron motif. The E2A-DBP degron expression plasmid was co-transfected with other plasmids expressing Rep / Cap, ITR-GOI, E4-E34K, and VA2. After 72 hours, cells were lysed, and AAV titer levels were analyzed. Cells transfected with the ITR-GOI plasmid alone (in which the helper and Rep / Cap plasmids were omitted to prevent AAV production) were used as negative control samples. Without the addition of the Shield1 molecule, AAV production is at the same level as the background levels of the qPCR results obtained from the negative control (solid red bar on the left and gray bar on the right). After addition of the Shieldl molecule, titer levels increased approximately 3-fold, demonstrating regulation of AAV production by degron-tagged E2A-DBP (striped middle band).Figure 19B shows Western blot results indicating a size shift in the E2A-DBP protein due to the addition of the degron tag. The tagged protein is -12 kDa larger than the unlabeled DBP protein. The two samples on the left represent unlabeled DBP, while the samples on the right represent DBP with the degron tag.

[0044] Figures 20A-20B show that a codon-modified Rep with a degron motif can be used to produce AAV. Figure 20A shows a modified rep gene under the control of a regulatory element, specifically the TRE3G-Tet system. The construct shown herein contains a large Rep protein produced only by the codon-modified Rep construct. Small Rep is expressed from a separate region of the same plasmid. Small rep is also under the control of TRE3G and a degron on the same plasmid. Cell samples with higher AAV titers were treated with Shieldl and Dox (Figure 20B). This demonstrates the inducible properties of multiple degron domains and Tet promoters on multiple AAV genes, including a modified rep gene, in the context of the methods disclosed herein.

[0045] 5. DETAILED DESCRIPTION

[0046] The currently disclosed subject matter relates to compositions and methods for regulating the production of recombinant rAAV in cell culture. In particular, the currently disclosed subject matter relates to compositions and methods for overcoming AAV Rep protein-mediated cytotoxicity by reversibly post-translationally regulating AAV Rep protein expression.

[0047] In one aspect, the subject matter of the present invention is directed to methods of culturing cells for post-translational regulation of rAAV expression. In some embodiments, reversible post-translational regulation of AAV Rep protein expression in cell culture is achieved by fusing a degradation domain to the AAV Rep protein. In some embodiments, fusing a degradation domain to the AAV Rep protein allows for regulated degradation of the AAV Rep protein based on the presence or absence of a degradation ligand in cell culture. In some embodiments, regulated degradation of the AAV Rep protein based on the presence or absence of a degradation ligand results in post-translational regulation of rAAV expression in cell culture.

[0048] In another aspect, the subject matter of the present invention is directed to rAAV-producing cells. For example, but not limited to, the present invention relates to rAAV-producing cells in which the expression of the AAV Rep protein is regulated by fusing a degradation domain to the AAV Rep protein.

[0049] In another aspect, reversible post-translational regulation of AAV helper protein expression in cell culture is achieved by fusing a degradation domain to the AAV helper protein. In some embodiments, fusing the degradation domain to the AAV helper protein allows for regulated degradation of the AAV helper protein based on the presence or absence of a degradation ligand in cell culture. In some embodiments, regulated degradation of the AAV helper protein based on the presence or absence of a degradation ligand results in post-translational regulation of rAAV expression in cell culture.

[0050] For clarity, but not limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections.

[0051] 5.1 Definitions

[0052] 5.2. Methods for regulating AAV expression using degradation domains

[0053] 5.3. Cells producing rAAV 5.1. Definitions

[0054] Terms used in this description generally have their ordinary meanings in the art, within the context of this disclosure, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the description to provide the practitioner with additional guidance in describing the compositions and methods of the present invention, as well as how to make and use them.

[0055] In this document, the use of a word indicating the singular when used in combination with the term "comprising" in the claims and / or description may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0056] The terms "comprise," "include," "having," "has," "may," "contain," and variations thereof, as used herein, are open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. This disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly set forth or not.

[0057] The term "approximately" or "approximately" means within an acceptable range of errors for a particular quantity, as determined by one skilled in the art, which will depend in part on how the quantity is measured or determined, i.e., the limitations of the measuring system. For example, "approximately" may mean within 3 or more than 3 standard deviations in accordance with the practice accepted herein. Alternatively, "approximately" may mean an interval of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given quantity. Alternatively, especially with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times the size, and more preferably within 2 times the size of the quantity.

[0058] In some embodiments, the cells of the present invention carry a chromosomally integrated rep gene but require helper virus function to express the Rep protein. "Helper virus" or "helper virus function" or "AAV helper" as used herein refers to at least one of adenovirus (Ad) E1 (e.g., E1a Ad or E1b Ad), E2A Ad, E4 Ad, and VA RNA, or the corresponding functions of other viruses, such as herpesviruses and poxviruses, that are capable of providing helper function to support the replication and packaging of AAV vector genomes. In specific embodiments, the hybrid virus consists of an adenovirus with an E1 / E3 deletion, but contains E2A Ad, E4 Ad, and VA RNA, which provide helper virus function, as well as AAV ITRs flanking a heterologous nucleic acid.In other embodiments, the hybrid viruses comprise the functions of a herpesvirus or poxvirus helper virus, as well as AAV ITRs flanking a heterologous nucleic acid.

[0059] As used herein, the term "helper virus function(s)" refers to the function(s) encoded in the helper virus genome that facilitates the replication and packaging of the rAAV vector genome (in combination with Rep and Cap). As disclosed herein, "helper virus function" can be provided in a number of different ways. For example, the helper virus function can be provided by the virus or, for example, provided by polynucleotide sequences encoding the necessary helper function(s) of the cell in trans. In another example, a plasmid or other expression vector containing polynucleotide sequences encoding one or more viral (e.g., adenoviral) proteins provides a helper function if, after transfection into a cell line of the invention, along with the rAAV vector genome, the replication and packaging of the rAAV vector genome into rAAV vector particles is ensured.In some embodiments, the helper virus function is provided by a virus selected from an adenovirus, a herpesvirus, a poxvirus, or a hybrid virus thereof. In some embodiments, the helper virus function comprises one or more viruses, vectors, or plasmids that provide the helper virus function. In some embodiments, the helper virus function comprises at least one of the Ad E1 protein (e.g., the Ad E1a protein or the Ad E1b protein), the Ad E2A protein, the Ad E4 protein, and the Ad VA RNA. In some embodiments, a degradation ligand-dependent degradation domain is fused to the helper protein.

[0060] As used herein, the term "AAV protein" refers to any wild-type or modified protein derived from the AAV genome and required for AAV production. "AAV protein" includes any form of Rep, Cap, or helper proteins, both wild-type and modified. Modifications to wild-type AAV genes need not result in a change in the amino acid sequence of the expressed protein. As used herein, the term "AAV fusion protein" refers to a fusion protein comprising an AAV protein, wherein the amino acid sequence of the AAV protein is fused directly or through a linker to other amino acid sequences, such as a degron sequence.

[0061] The term "vector" refers to a small molecule nucleic acid carrier, such as a plasmid, virus (e.g., AAV vector), or other carrier, that can be manipulated by inserting or incorporating nucleic acid. Such vectors can be used for genetic manipulation (i.e., "cloning vectors"), for introducing / transferring polynucleotides into cells, and for transcription or translation of the inserted polynucleotides into cells. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions required for expression in a host cell.

[0062] The nucleic acid sequence of the vector typically comprises at least an origin of replication for propagation in the cell and optionally additional elements such as a heterologous polynucleotide sequence, an expression control element (e.g., a promoter, an enhancer), an intron, an inverted terminal repeat (ITR), a selectable marker (e.g., antibiotic resistance), a polyadenylation signal.

[0063] A viral vector is derived from or based on one or more nucleic acid elements that make up the viral genome. A specific viral vector is the adeno-associated virus (AAV) vector.

[0064] The term "recombinant" as a modifier of a vector, such as a recombinant AAV vector, as well as a modifier of sequences, such as recombinant polynucleotides and polypeptides, means that the compositions have been processed (i.e., constructed) in a manner that is not typically found in nature. A specific example of a recombinant AAV vector would be a click acid sequence that is not typically present in the wild-type AAV genome (e.g., a heterologous nucleic acid sequence) inserted into the AAV genome. Although the term "recombinant" is not always used herein with respect to AAV vectors and sequences, such as polynucleotides, recombinant forms, including polynucleotides, are expressly included despite any such omission.

[0065] A "recombinant AAV vector" or "rAAV" is derived from a wild-type (WT or wild-type) AAV genome using molecular techniques to remove the wild-type genome from the AAV genome and replace it with a non-native nucleic acid sequence, referred to as a heterologous nucleic acid sequence. Typically for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the AAV vector. rAAV differs from the AAV genome because all or part of the AAV genome is replaced with a non-native sequence relative to the AAV genomic nucleic acid. Thus, the inclusion of a non-native sequence defines the AAV vector as a "recombinant" vector, which may be referred to as an "rAAV vector."

[0066] The rAAV sequence can be packaged, referred to herein as a "particle," for subsequent cell transduction ex vivo, in vitro, or in vivo. If the recombinant AAV vector sequence is encapsidated or packaged within an AAV particle, the particle may also be referred to as an "rAAV vector" or "rAAV particle." Such rAAV particles include proteins that encapsidate or package the vector genome. In the case of AAV, these are called capsid proteins.

[0067] The "genome" of a vector refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral particle (e.g., AAV). When recombinant plasmids are used to construct or produce recombinant vectors, the vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid genome is called the "plasmid backbone," which is important for plasmid cloning and amplification, a process required for the propagation and production of the recombinant virus, but is not itself packaged or encapsidated into viral particles (e.g., AAV). Thus, the vector "genome" refers to the nucleic acid that is packaged or encapsidated by the virus (e.g., AAV).

[0068] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and

[0069] Ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA-tRNA, and inhibitory DNA or RNA (RNAi, such as small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., a nucleic acid variant). Nucleic acids such as cDNA, genomic DNA, RNA, and fragments thereof, which may be single- or double-stranded.

[0070] Polynucleotides may be single, double, or triple, linear or circular, and may be of any length. When discussing polynucleotides in this document, the sequence or structure of a particular polynucleotide may be described according to the generally accepted sequence representation in the 5' to 3' direction.

[0071] The term "transgene" is used herein to refer to a heterologous nucleic acid that is intended for, or has been introduced into, a cell or organism. Transgenes include any heterologous nucleic acid, such as a gene that encodes a polypeptide or protein or encodes inhibitory RNA.

[0072] Heterologous nucleic acid can be introduced / transferred into a cell using a vector such as AAV, "transduction," or "transfection." The term "transduce" and its grammatical variations refer to the introduction of a molecule, such as an rAAV vector, into a host cell or organism. The introduced heterologous nucleic acid may also exist in the recipient cell or host organism extrachromosomally or only transiently.

[0073] A "transduced cell" is a cell into which a transgene has been introduced. Accordingly, a "transduced" cell (e.g., in a mammal, such as a cell or a cell of a tissue or organ) means a genetic change in the cell following the incorporation of an exogenous molecule, such as a nucleic acid (e.g., a transgene) into the cell. Thus, a "transduced" cell is a cell or its progeny into which an exogenous nucleic acid has been introduced. The cell(s) may

[0074] (can) be expanded and the introduced protein expressed or nucleic acid transcribed. For gene therapy applications and methods, the transduced cell may reside in the subject's body.

[0075] An "expression control element" is a type of regulatory element and refers to nucleic acid sequence(s) that influence the expression of an operably linked nucleic acid. Control elements, including expression control elements as defined herein, such as promoters and enhancers. Vector sequences, including AAV vectors, may include one or more "expression control elements." Typically, such elements are included to facilitate the proper transcription of heterologous polynucleotides and, if necessary, translation (e.g., a promoter, enhancer, splicing signal for introns, maintenance of the correct gene reading frame to ensure in-frame translation of mRNA and stop codons, etc.). Such elements typically act in the cis position, referred to as a "cis-acting" element, but may also act in the trans position.

[0076] Expression control can occur at the level of transcription, translation, splicing, message stability, and so on. Typically, an expression control element that modulates transcription is located near the 5' end (i.e., "upstream") of the transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron).

[0077] Functionally, expression of an operably linked nucleic acid is at least partially controlled by an element (e.g., a promoter), such that the element modulates transcription of the nucleic acid and, if necessary, translation of the transcript. A specific example of an expression control element is a promoter, which is typically located at the 5' end of the nucleic acid sequence being transcribed. A promoter typically increases the amount of the operably linked nucleic acid expressed compared to the amount expressed in the absence of the promoter.

[0078] Another type of regulatory element involves Tet. In Tet-dependent induction, expression of the target transgene depends on an inducible promoter. The promoter can be regulated by levels of tetracycline or tetracycline derivatives, such as doxycycline (Dox). Activation of Tet-On promoters depends on the presence of an additional activator protein, which can bind to promoters in the presence of Dox. In contrast, transcription is inactive in the presence of Dox for the Tet-Off system. Other examples of inducible systems include cumate, abscisic acid (ABA), rapamycin, and tamoxifen-inducible systems. The degradation ligand-dependent degradation domain described herein can also be used with Cre-LoxP, CRISPR, riboswitches, and light-switchable transgene systems. The term "enhancer" as used herein may refer to a sequence located adjacent to a heterologous nucleic acid.Enhancer elements are typically located upstream of a promoter element, but they also function and can be located downstream or within the sequence. Enhancer elements typically increase the expression of the functionally linked nucleic acid compared to that mediated by the promoter element.

[0079] In this document, expression control elements such as promoters are typically located at a distance from the transcribed sequence.In particular embodiments, an expression control element, such as a promoter, is located at least about 25 nucleotides from the 5' end of the rep gene start codon, located about 25-5000 nucleotides from the 5' end of the rep gene start codon, located about 250-2500 nucleotides from the 5' end of the rep gene start codon, located about 500-2000 nucleotides from the 5' end of the rep gene start codon, located about 1000-1900 nucleotides from the 5' end of the rep gene start codon, located about 1500-1900 nucleotides from the 5' end of the rep gene start codon, located about 1600-1800 nucleotides from the 5' end of the rep gene start codon, located about 1700-1800 nucleotides from 5'-end of the start codon of the rep gene or located approximately 1750 nucleotides from the 5'-end of the start codon of the rep gene.

[0080] Expression control elements include ubiquitous or promiscuous promoters / enhancers that are capable of driving expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter / enhancer sequences, the Rous sarcoma virus (RSV) promoter / enhancer sequences and other viral promoters / enhancers active in various mammalian cell types or synthetic elements (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, and the phosphoglycerol kinase (PGK) promoter.

[0081] Expression control elements also include native elements of the heterologous polynucleotide. A native control element (e.g., a promoter) can be used when it is desired that expression of the heterologous polynucleotide mimic native expression. Other native control elements, such as introns, polyadenylation sites, or Kozak consensus sequences, can also be used.

[0082] The term "operably linked" means that the regulatory sequences required for expression of a nucleic acid sequence are placed at appropriate positions relative to the sequence to ensure expression of the nucleic acid sequence. This same definition is sometimes applied to the arrangement of nucleic acid sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector, such as the rAAV vector.

[0083] In an example of an expression control element functionally linked to a nucleic acid, the relationship is such that the control element modulates the expression of the nucleic acid. More specifically, for example, two functionally linked DNA sequences mean that the two DNA sequences are arranged (in cis or trans) in a relationship such that at least one of the DNA sequences is capable of exerting a physiological effect on the other sequence.

[0084] As disclosed herein, a nucleic acid spacer sequence located between the expression control element and the AAV rep gene can significantly reduce or eliminate the expression of the rep gene, thereby reducing or eliminating the expression of the Rep protein and allowing cells to survive, even if the cells also express the adenovirus E1a protein. Addition of helper virus function to such cells, such as that provided by a hybrid virus, adenovirus, poxvirus, or herpesvirus, can overcome the attenuating effect of the spacer nucleic acid on rep gene expression and, in turn, stimulate the expression of the rep gene, thereby ensuring the expression of the Rep protein.

[0085] Additional elements for rAAV vectors include, but are not limited to, a transcription termination signal or stop codon, 5'- or 3'-untranslated regions (e.g., polyadenylation (polyA) sequences) that flank the sequence, such as one or more copies of an AAV ITR sequence or an intron.

[0086] Additional elements include, for example, filler or spacer polynucleotide sequences, e.g., to improve packaging and reduce the presence of contaminating nucleic acid. AAV vectors typically accommodate DNA inserts that are typically between approximately 4 and approximately 5.2 kb in size or slightly larger. Thus, for shorter sequences, the inclusion of a spacer or filler is necessary to bring the length to a level close to the normal size of the viral genomic sequence, acceptable for packaging the AAV vector into a viral particle. In various embodiments, the filler / spacer nucleic acid sequence is an untranslated (non-protein-coding) segment of nucleic acid. For a nucleic acid sequence of less than 4.7 kb in size,the polynucleotide sequence of the filler or spacer has a length that, when combined (e.g., inserted into a vector) with the sequence, has a total length of approximately 3.0-5.5 kb, or approximately 4.0-5.0 kb, or approximately 4.3-4.8 kb.

[0087] If a heterologous nucleic acid or wild-type transgene is too large to be packaged into an AAV vector particle, the heterologous nucleic acid may be provided in a modified, fragmented, or truncated form for packaging and delivery by the AAV vector, so that a functional protein or nucleic acid product, such as a therapeutic protein or nucleic acid product, is ultimately provided.

[0088] In some embodiments, the heterologous nucleic acid that encodes a protein (e.g., a therapeutic protein) is provided in a modified or truncated form, or the heterologous nucleic acid is provided as a plurality of constructs delivered by separate and multiple AAV vectors.

[0089] In some aspects, the heterologous nucleic acid is provided as a truncated version that retains the functionality of the encoded protein (e.g., a therapeutic protein), including the removal of portions unnecessary for function, such that the encoding heterologous polynucleotide is reduced in size for packaging into an AAV vector.

[0090] In some aspects, the heterologous nucleic acid is provided in separate AAV vectors, each of which contains nucleic acid encoding different portions of a protein (e.g., a therapeutic protein), thereby delivering multiple portions of a protein (e.g., a therapeutic protein) that assemble and function in a cell.

[0091] In other aspects, the heterologous nucleic acid is represented by dual AAV vectors using overlapping, trans-splicing, or hybrid dual trans-splicing vector technology. In some embodiments, two overlapping AAV vectors are used that are combined in a cell to create a complete expression cassette from which a full-length protein (e.g., a therapeutic protein) is expressed.

[0092] “Disorder related to hemostasis” refers to bleeding disorders such as hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any of the coagulation factors VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency or gamma-carboxylase deficiency; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy or disseminated intravascular coagulation (DIC);

[0093] excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, or low molecular weight antithrombotic agents (i.e., FXa inhibitors); and platelet disorders such as Bernard Soulier syndrome, Glanzmann thrombasthenia, and storage pool deficiency.

[0094] The term "isolated," when used as a composition modifier, means that the compositions are manufactured by human hands or separated, in whole or at least in part, from their natural environment in vivo. Typically, isolated compositions are substantially free of one or more materials with which they are normally associated in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, or cell membranes.

[0095] The term "isolated" does not exclude combinations created by human hands, such as an rAAV sequence or an rAAV particle that packages or encapsidates an AAV vector genome and a pharmaceutical composition. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrids / chimeras, multimers / oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation), or derivatized forms, or forms expressed in host cells produced by human hands.

[0096] The term "substantially pure" refers to a preparation containing at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation may contain at least 75% by weight, or at least 85% by weight, or approximately 90-99% by weight of the compound of interest. Purity is measured by methods suitable for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0097] The phrase "consisting essentially of," when used with respect to a specific nucleotide sequence or amino acid sequence, means a sequence having the properties of that SEQ ID NO. For example, when used with respect to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the basic and novel characteristics of the sequence.

[0098] The terms "identity," "homology," and their grammatical variants mean that two or more referenced entities are the same when they represent "aligned" sequences. For example, when two protein sequences are identical, they have the same amino acid sequence, at least within the specified region or portion. When two nucleic acid sequences are identical, they have the same nucleic acid sequence, at least within the specified region or portion. Identity may extend to a specific region (region or domain) of the sequence.

[0099] A "region" or "section" of identity refers to the portion of two or more specified entities that are the same. Thus, if two protein or nucleic acid sequences are identical in one or more regions or sections of the sequence, they share overall identity within that region. An "aligned" sequence refers to multiple protein (amino acid) or nucleic acid sequences, often containing corrections for missing or additional bases or amino acids (gaps) compared to a reference sequence.

[0100] The identity may extend over the entire length or a portion of the sequence. In some embodiments, the length of the sequence having percentage identity is 2, 3, 4, 5 or more contiguous amino acids or nucleic acids, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. contiguous nucleic acids or amino acids. In further embodiments, the length of the sequence having identity is 21 or more contiguous amino acids or nucleic acids, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc. contiguous amino acids or nucleic acids. In further embodiments, the length of the sequence having identity is 41 or more contiguous amino acids or nucleic acids, such as 42, 43, 44, 45, 45, 47, 48, 49, 50, etc. contiguous amino acids or nucleic acids.In further embodiments, the length of the sequence having identity is 50 or more contiguous amino acids or nucleic acids, such as 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-150, 150-200, 200-250, 250-300, 300-500, 500-1000, etc. contiguous amino acids or nucleic acids.

[0101] The degree of identity (homology) or "percent identity" between two sequences can be determined using a computer program and / or a mathematical algorithm. For the purposes of this invention, nucleic acid sequence comparisons are performed using the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wisconsin. For convenience, the default parameters (gap creation penalty=12, gap extension penalty=4) specified by this program are intended for use herein for sequence identity comparisons. Alternatively, the Blastn 2.0 program provided by the National Center for Biotechnology Information (available on the World Wide Web at ncbi.nlm.nih.gov / blast / ; Altschul et al., 1 990, J Mol Biol 215:403–410) using gapped alignment with default parameters, can be used to determine the level of identity and similarity between nucleic acid sequences and amino acid sequences. For comparison of polypeptide sequences, the BLASTP algorithm is typically used in conjunction with a scoring matrix such as PAM100, PAM 250, BLOSUM 62, or BLOSUM 50. The sequence comparison programs FASTA (e.g., FASTA2 and FASTA3) and SSEARCH are also used to quantify the degree of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2 4 4 4 (198 8); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs have also been developed to quantify the structural similarity of proteins using Delaunay-based topology mapping (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0102] Nucleic acid molecules, expression vectors (e.g., AAV vector genomes), plasmids, including nucleic acid encoding modified / variant AAV capsids of the invention, and heterologous nucleic acids can be produced using recombinant DNA technology. The availability of nucleotide sequence information allows for the production of isolated nucleic acid molecules of the invention in a variety of ways. For example, nucleic acid sequences can be obtained using various standard cloning technologies, recombinant DNA, through cellular expression, or in vitro translation and chemical synthesis methods. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, etc. For example, nucleic acids can be isolated using hybridization or computer database screening methods.Such methods include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides sharing common structural features, such as using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of hybridizing to the nucleic acid sequence of interest; (4) computer searches for related sequences in sequence databases; and (5) differential screening of a subtractive nucleic acid library.

[0103] 5.2. Methods for regulating AAV production using degradation domains In one aspect, the present invention is directed to methods of culturing cells for post-translational regulation of rAAV expression. In some embodiments, reversible post-translational regulation of AAV Rep protein expression in cell culture is achieved by fusing a degradation domain ("degron") to the AAV Rep protein. In some embodiments, fusing a degradation domain to the AAV Rep protein allows for regulation of the degradation of the AAV Rep protein based on the presence or absence of a degradation ligand or other "inhibitor," such as a small molecule ligand that binds to a degron to alter its rate of degradation or an inhibitor, such as light or temperature, that alters the rate of degron degradation.In some embodiments, regulated degradation of the AAV Rep protein based on the presence or absence of a degradation ligand or other inhibitor results in post-translational regulation of rAAV expression in cell culture. As used herein, the term "degradation ligand-dependent degradation domain" refers to a degron domain that binds a degradation ligand. As used herein, the term "degradation ligand" refers to a ligand that binds a degradation domain.

[0104] Any suitable degron can be used in connection with the methods of the present invention. For example, but not limited to, the degron is modified from a human gene encoding a protein called FK506-binding protein 12 ("FKBP"). In some embodiments, the degron is derived from FKBP. In some embodiments, the FKBP protein variant from which the FKBP degron is derived comprises an F3 6V amino acid substitution. In some embodiments, the FKBP protein variant from which the FKBP degron is derived comprises an L106P amino acid substitution. In some embodiments, the FKBP protein variant from which the FKBP degron is derived comprises both an F36V and an L106P amino acid substitution. In some embodiments, the FKBP modifications: a) deepen the binding pocket to improve its specificity for a degradation ligand, e.g., Shield1, compared to FK506; and / or b) make the protein more unstable in the absence of its degradation ligand.dTAG-13 is a small molecule that can target mutant sequences of FKBP12 (F36V) for ubiquitin-mediated degradation. It can function by linking the target protein sequence to the E3 ubiquitin ligase, cereblon. dTAG-13 can lead to the degradation of FKBP12-F36V fusion proteins and proteins fused to it. For the purposes of this document, the FKBP12 domain is referred to as "FKBP."

[0105] In some embodiments, the degron is a dihydrofolate reductase (DHFR)-based degron; an auxin-induced degron domain (AID); an ornithine decarboxylase (ODC)-based degron; a cleaved ubiquitin-based degron system; a protease-based degron system; a proteolysis-targeted-chimeric molecule (PROTAC)-based degron system; an antibody-dependent protein degron system; a light-sensitive degron (psd); a phosphorylation-dependent degron; or a temperature-dependent degron.

[0106] In some embodiments, the degron is regulated by the presence or absence of a degradation ligand or inhibitor. In some embodiments, the degradation ligand is a small molecule ligand. In some embodiments, for example, if the degron is a variant of the FKBP protein, the small molecule ligand is Shield1.

[0107] In some embodiments, for example, if the degron is a dihydrofolate reductase (DHFR)-based degron, the ligand is trimethoprim (TMP). In some embodiments, for example, if the degron is an auxin-induced degron domain (AID), the ligand is auxin. In some embodiments, for example, if the degron is an ornithine decarboxylase (ODC)-based degron, the ligand is antizyme. In some embodiments, for example, if the degron is a cleaved ubiquitin-based degron system, the ligand is rapamycin. In some embodiments, for example, if the degron is a protease-based degron system, the inhibitor can be an inhibitor of HCV protease or TEV protease expression.In some embodiments, for example, if the degron is a proteolysis-targeted chimeric degron system (PROTAC), the inhibitor is the expression of the PROTAC. In some embodiments, for example, if the degron is an antibody-dependent protein degron system, the ligand is the corresponding antibody. In some embodiments, for example, if the degron is a light-sensitive degron (psd), the inhibitor is light. In some embodiments, for example, if the degron is a phosphorylation-dependent degron, the inhibitor is the corresponding kinase activator. In some embodiments, for example, if the degron is a temperature-dependent degron, the inhibitor is a change in temperature.

[0108] In some embodiments, the degron sequence is linked to the C-terminus of the AAV Rep protein. In some embodiments, the degron sequence is linked to the N-terminus of the AAV Rep protein. In some embodiments, the degron and the AAV Rep protein are linked via a flexible linker. In some embodiments, the degron and the AAV Rep protein are linked via a rigid linker. In some non-limiting embodiments, the flexible linker has the amino acid sequence: GGGGSGGGGSGGGGS. In some non-limiting embodiments, the rigid linker has the amino acid sequence:

[0109] AEAAAKEAAAKEAAAKEAAAKALEEAAAKEAAAKEAAAKEA AAKA.

[0110] In some embodiments, the methods of the present invention relate to regulating the production of a recombinant adeno-associated virus (rAAV), wherein the method comprises: introducing into a mammalian cell an rAAV containing a gene of interest and a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a Rep protein, a linker, and a degradation domain dependent on a degradation ligand, culturing the cell under conditions suitable for producing the rAAV virus; and contacting the cell with a degradation ligand, wherein the degradation ligand binds to the degradation domain, regulating the expression of the Rep protein and thereby regulating the production of rAAV.

[0111] In some embodiments, the cell is a cell expressing E1a. In some embodiments, the cell is a human cell. In some embodiments, the cell is a HEK293 cell. In some embodiments, the cell is a HEK293F cell. In some embodiments, the cell is a PERC6 cell.

[0112] In some embodiments, the Rep protein is a Rep78, Rep68, Rep52, or Rep40 protein.

[0113] The methods of the present invention include the use of suitable regulatory elements, including promoters, to drive the expression of Rep and Cap proteins. In some embodiments, suitable promoters can be eukaryotic, prokaryotic, or viral promoters. Suitable promoters include non-inducible promoters and non-tissue-specific promoters. In some embodiments, the promoter is the AAV p5 promoter, which in its native state drives the expression of the Rep protein from the rep gene. In some embodiments, the promoter is the cytomegalovirus (CMV) immediate-early promoter / enhancer. Additional non-limiting examples of suitable promoters include ubiquitous or promiscuous promoters / enhancers that are capable of driving the expression of a polynucleotide in many different cell types.Such elements include, but are not limited to, Rous sarcoma virus (RSV) promoter sequences and other viral promoters active in various mammalian cell types, or synthetic elements (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic b-actin promoter, and the phosphoglycerol kinase (PGK) promoter. In some embodiments, the promoter is selected from the human alpha-1 elongation factor (laEF1) promoter, the CAG promoter, the CBA promoter, the SFFV promoter, the p19 promoter, and the herpes simplex virus thymidine kinase (HSV-TK) promoter. In some embodiments, promoters may be located proximal or distal to the rep and cap genes. In some embodiments, regulatory elements, including promoters, may be in cis or trans relative to the rep and cap genes.In some embodiments, the methods of the present invention are directed to expressing a nucleic acid encoding an AAV Rep protein, wherein the AAV Rep protein is an AAV1 Rep protein, an AAV2 Rep protein, an AAV3 Rep protein, an AAV4 Rep protein, an AAV5 Rep protein, an AAV6 Rep protein, an AAV7 Rep protein, an AAV8 Rep protein, an AAV9 Rep protein, an AAV 10 Rep protein, or an AAV11 Rep protein. Since the methods of the present invention are applicable to any type of cell that produces an AAV, the AAV may be of, for example, human, avian, bovine, canine, equine, primate, non-primate, ovine origin, or any derivative thereof.

[0114] In some embodiments, the rAAV produced by the methods of the present invention includes any viral strain or serotype. In some non-limiting embodiments, the rAAV may be based on any AAV genome, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, Rh8, RhlO, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80 and variants thereof, including the AAV capsid variants set forth in Pulicherla et al., Mol. Ther., 19(6) 1070–1078 (2011) (description of AAV9 variants, including, inter alia, AAV9.47), U.S. Patent No. 7,906,111 (describing, inter alia, AAV9(hul4)), U.S. Patent No. 1,0532,111 (describing, inter alia, NP59), U.S. Patent No. US1,0738,087 (describing, inter alia, Anc-80), WO2012 / 145601, WO2013 / 158879, WO2015 / 013313, WO2018 / 156654, US2013 / 0059732, U.S. Patent Nos. 9,169,299 (describing LK03), 9,840,719 (describing RHM4-1), 77 4 94 92, 75887 72 (describing DJ and DJ8) and 9587282, all of which are incorporated herein by reference in their entirety. Therefore, rAAV vectors include gene / protein sequences identical to those characteristic of a specific serotype, as well as those of mixed serotypes.

[0115] In some embodiments, the nucleic acid encoding the AAV Rep and Cap proteins is arranged in the same manner as in the native AAV genome. In some embodiments, the nucleic acid encoding the AAV Rep and Cap proteins is arranged in the same manner as in the native AAV genome, except that the nucleic acid encodes a linker and / or a degron sequence at the N-terminus or C-terminus of the AAV Rep protein. In some embodiments, the nucleic acids encoding the AAV Rep and Cap proteins are not arranged directly or indirectly in a tandem configuration. In some embodiments, the nucleic acids encoding the AAV Rep and Cap proteins are not covalently linked.Additional suitable arrangements of the AAV Rep and Cap coding sequences may be used, for example, the order of the AAV Rep and Cap coding sequences may be reversed relative to their natural AAV genomic order, one or both of the AAV Rep and Cap coding sequences may be preceded or followed by a sequence containing an IRES, a sequence encoding a self-cleaving protein (e.g., peptide 2A), or a spacer region that has no function. In some embodiments, one or both of the AAV Rep and Cap coding sequences may be included in the genome of the cell. In some embodiments, one or both of the AAV Rep and Cap coding sequences may be combined with factors that increase the amount of DNA sequences that enhance the maintenance of episomal plasmids.

[0116] In some embodiments, the helper virus function is provided by a virus selected from an adenovirus, a herpesvirus, a poxvirus, or a hybrid virus thereof. In some embodiments, the helper virus function comprises one or more viruses, vectors, or plasmids that provide the helper virus function. In some embodiments, the helper virus function comprises at least one of adenovirus (Ad) E1 protein (e.g., Ad E1a or Ad E1b protein), Ad E2A protein, Ad E4 protein, and Ad VA RNA.

[0117] The function of a helper virus can be mediated in a number of different ways. In some embodiments, the function of a helper virus can be mediated by the virus or, for example, by polynucleotide sequences encoding the necessary helper function(s) of the cell in trans.

[0118] In some embodiments, the degron sequence is linked to the C-terminus of an adenovirus (Ad) E1 protein (e.g., an Ad E1a or Ad E1b protein), an Ad E2A protein, or an Ad E4 protein. In some embodiments, the degron sequence is linked to the C-terminus of an Ad E1 protein (e.g., an Ad E1a or Ad E1b protein), an Ad E2A protein, or an Ad E4 protein. In some embodiments, the degron and the Ad E1 protein (e.g., an Ad E1a or Ad E1b protein), an Ad E2A protein, or an Ad E4 protein are linked via a flexible linker. In some embodiments, the degron and the Ad E1 protein (e.g., an Ad E1a or Ad E1b protein), an Ad E2A protein, or an Ad E4 protein are linked via a rigid linker.

[0119] In some embodiments, two or more AAV proteins, such as Rep, Cap, and / or helper proteins, are independently linked to a degron sequence to form two or more AAV-protein-degron fusion proteins. In some embodiments, each of the two or more AAV-protein-degron fusion proteins comprises a degron at the C-terminus or N-terminus of the AAV protein. In some embodiments, each of the two or more AAV-protein-degron fusion proteins comprises a different degron sequence. In some embodiments, each of the two or more AAV-protein-degron fusion proteins comprises the same degron sequence. In some embodiments, the linkage between the AAV protein and the corresponding degron is a flexible linker. In some embodiments, the linkage between the AAV protein and the corresponding degron is a rigid linker.

[0120] In some embodiments, two or more AAV-protein fusion proteins may be encoded by sequences of the same vector, such as a plasmid. In some embodiments, two or more AAV-protein-degron fusion proteins may be encoded by sequences of separate vectors, such as a first plasmid comprising a coding sequence for a first AAV-protein-degron fusion protein, and a second plasmid comprising a coding sequence for a second AAV-protein-degron fusion protein. In some embodiments, expression of at least one AAV-protein-degron fusion protein is under the control of a regulatory element. In some embodiments, expression of at least two AAV-protein-degron fusion proteins is under the control of a regulatory element. In some embodiments, expression of multiple AAV-protein-degron fusion proteins is under the control of different regulatory elements.In some embodiments, expression of all AAV-protein-degron fusion proteins is under the control of a regulatory element. In some embodiments, the regulatory element is a promoter. In some embodiments, the regulatory element is a Tet response element.

[0121] In some embodiments, a cell of the present invention comprises an acid sequence that is not normally present in the wild-type AAV genome, such as a heterologous nucleic acid sequence, also referred to herein as a gene of interest or (GOI). In some non-limiting embodiments, the GOI comprises a nucleic acid sequence encoding a therapeutic protein or an inhibitory nucleic acid sequence. In some embodiments, the GOI can be introduced / transferred into the cell using a vector, such as AAV transduction or transfection. In some embodiments, the introduced GOI can also exist in the recipient cell or host organism extrachromosomally or only transiently.

[0122] In some embodiments, the GOI encodes a protein (e.g., a therapeutic protein) that is present in a modified or truncated form, or the GOI is present as a plurality of constructs delivered by separate and multiple AAV vectors.

[0123] In some embodiments, the GOI is provided as a truncated version that retains the functionality of the encoded protein (e.g., a therapeutic protein), including the removal of portions unnecessary for function, such that the GOI is reduced in size for packaging into an AAV vector.

[0124] In some embodiments, the GOI is provided in separate AAV vectors, each of which contains nucleic acid encoding different portions of a protein (e.g., a therapeutic protein), thereby delivering multiple portions of a protein (e.g., a therapeutic protein) that assemble and function in a cell.

[0125] In some embodiments, the GOI is represented by dual AAV vectors using overlapping, trans-splicing, or hybrid dual trans-splicing vector technology. In some embodiments, two overlapping AAV vectors are used that are combined in the cell to create a complete expression cassette from which a full-length protein (e.g., a therapeutic protein) is expressed.

[0126] 5.3. AAV-producing cells

[0127] In another aspect, the subject matter of the present invention is directed to rAAV-producing cells. For example, but not limited to, the present invention relates to rAAV-producing cells in which the expression of the AAV Rep protein is regulated by fusing a degradation domain to the AAV Rep protein.

[0128] Embodiments of rAAV-producing cells of the present invention include any cell type or system that can produce rAAV or AAV. Several examples of various systems have been previously described, for example, Conlon and Mavilio, Mol. Therapy, 8:181-182 (2018), the entire contents of which are incorporated herein by reference. Embodiments of the present invention include rAAV production by transient transfection of plasmids into mammalian cells, rAAV production in stable cell lines, rAAV production by herpes simplex virus in mammalian cells, and rAAV production by baculovirus in Sf9 cells.

[0129] In some embodiments, the rAAV-producing cells of the present invention are eukaryotic cells. In some embodiments, the rAAV-producing cells of the present invention are animal cells. In some embodiments, the rAAV-producing cells of the present invention are insect cells. In some embodiments, the rAAV-producing cells of the present invention are mammalian cells. In some embodiments, the rAAV-producing cells of the present invention are human cells. In some embodiments, the rAAV-producing cells of the present invention are human embryonic kidney (HEK) cells. In some embodiments, the rAAV-producing cells of the present invention are HEK293 cells, HEK293F cells, or Expi293 cells.

[0130] In some embodiments, the rAAV-producing cells of the present invention are Chinese hamster ovary (CHO) cells.

[0131] In some embodiments, the rAAV-producing cells of the present invention are insect cells (Sf9).

[0132] In some embodiments, the rAAV-producing cells of the present invention do not express the SV40 large T antigen.

[0133] In some embodiments, the rAAV-producing cells of the present invention are suspension cells. In some embodiments, the rAAV-producing cells of the present invention are adherent cells.

[0134] In some embodiments, rAAV-producing cells of the present invention may be cultured at a cell density of at least about 1×10 6 , at least approximately 5×10 6, at least approximately 1×10 7 or at least approximately 2×10 7 cells / ml. In some embodiments, rAAV-producing cells of the present invention can be cultured at a cell density of about 1×10 6 up to 5×10 6 , approximately 5×10 6 up to 1×10 7 or approximately 1×10 7 up to 2×10 7 cells / ml.

[0135] In some embodiments, the rAAV-producing cells of the present invention are present in a culture or growth medium.

[0136] In some embodiments, the rAAV-producing cells of the present invention are in a medium suitable for storage. In some embodiments, the rAAV-producing cells of the present invention are in a medium suitable for long-term storage at a temperature of 0°C or lower, -30°C or lower, -80°C or lower, or -160°C or lower.

[0137] In some embodiments, the present invention relates to a mammalian cell producing rAAV, wherein the cell comprises a nucleic acid encoding a fusion protein comprising a Rep protein, a linker, and a degradation domain dependent on a degradation ligand. In some embodiments, the degradation domain dependent on a degradation ligand is a variant of the FKBP protein. In some embodiments, the degradation ligand is a small molecule ligand. In some embodiments, the small molecule is Shield1.

[0138] In some embodiments, the mammalian cell producing rAAV of the present invention is a cell expressing E1a. In some embodiments, the cell expressing E1a is a HEK293 cell.

[0139] In some embodiments, the degradation ligand-dependent degradation domain of the rAAV-producing mammalian cell of the present invention is fused via a linker to the C-terminal end of the Rep protein. In some embodiments, the degradation ligand-dependent degradation domain of the rAAV-producing mammalian cell of the present invention is fused via a linker to the N-terminal end of the Rep protein. In some embodiments, the linker is a flexible linker. In some non-limiting embodiments, the flexible linker has the amino acid sequence: GGGGSGGGGSGGGGS. In some embodiments, the linker is a rigid linker. In some non-limiting embodiments, the rigid linker has the amino acid sequence: AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAK EAAAKEAAAAKA.

[0140] In some embodiments, a mammalian cell producing rAAV according to the present invention comprises a nucleic acid encoding the AAV Rep and Cap proteins arranged in the same manner as in the native AAV genome. In some embodiments, the nucleic acid encoding the AAV Rep and Cap proteins is arranged in the same manner as in the native AAV genome, except that the nucleic acid encodes a linker and / or a degron sequence at the N-terminus or C-terminus of the AAV Rep protein. In some embodiments, the nucleic acids encoding the AAV Rep and Cap proteins are not arranged directly or indirectly in a tandem configuration. In some embodiments, the nucleic acids encoding the AAV Rep and Cap proteins are not covalently linked.Additional suitable arrangements of the AAV Rep and Cap coding sequences may be used, for example, the order of the AAV Rep and Cap coding sequences may be reversed relative to their natural AAV genomic order, one or both of the AAV Rep and Cap coding sequences may be preceded or followed by a sequence containing an IRES, a sequence encoding a self-cleaving protein (e.g., peptide 2A), or a spacer region that has no function. In some embodiments, one or both of the AAV Rep and Cap coding sequences may be included in the genome of the cell. In some embodiments, one or both of the AAV Rep and Cap coding sequences may be combined with factors that increase the amount of DNA sequences that enhance the maintenance of episomal plasmids.

[0141] In some embodiments, a mammalian cell producing rAAV according to the present invention comprises a virus capable of performing the function of a helper virus. In some embodiments, the virus is selected from an adenovirus, a herpesvirus, a poxvirus, or a hybrid virus thereof. In some embodiments, a mammalian cell producing rAAV according to the present invention comprises one or more viruses, vectors, or plasmids that provide the function of a helper virus. In some embodiments, the function of a helper virus includes at least one of an adenovirus (Ad) E1 protein (e.g., an Ad E1a or Ad E1b protein), an Ad E2A protein, an Ad E4 protein, and an Ad VA RNA.

[0142] In some embodiments, a mammalian cell producing rAAV according to the present invention comprises a GOI. In some non-limiting embodiments, the GOI comprises a nucleic acid sequence encoding a therapeutic protein or an inhibitory nucleic acid sequence. In some embodiments, the GOI can be introduced / transferred into the cell using a vector, such as AAV transduction or transfection. In some embodiments, the introduced GOI can also exist in the recipient cell or host organism extrachromosomally or only transiently.

[0143] In some embodiments, the GOI encodes a protein (e.g., a therapeutic protein) that is present in a modified or truncated form, or the GOI is present as a plurality of constructs delivered by separate and multiple AAV vectors.

[0144] In some embodiments, the GOI is provided as a truncated version that retains the functionality of the encoded protein (e.g., a therapeutic protein), including the removal of portions unnecessary for function, such that the GOI is reduced in size for packaging into an AAV vector.

[0145] In some embodiments, the GOI is provided in separate AAV vectors, each of which contains a nucleic acid encoding different portions of a protein (e.g., a therapeutic protein), thereby delivering multiple portions of a protein (e.g., a therapeutic protein) that assemble and function in a mammalian cell producing rAAV according to the present invention.

[0146] In some embodiments, the GOI is represented by dual AAV vectors using overlapping, trans-splicing, or hybrid dual trans-splicing vector technology. In some embodiments, two overlapping AAV vectors are used that are combined in a mammalian cell producing rAAV according to the present invention to create a complete expression cassette from which a full-length protein (e.g., a therapeutic protein) is expressed.

[0147] Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) that are useful according to the invention include those that can be used in the treatment of a disease or disorder, including, but not limited to, "hemostasis" or bleeding disorders, such as hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, deficiencies of coagulation factors VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor, combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase CI deficiency, gamma-carboxylase deficiency; anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation syndrome (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, low molecular weight antithrombotic agents (i.e.FXa inhibitors); and platelet disorders such as Bernard-Soulier syndrome, Glanzmann thrombocytopenia, and platelet pool deficiency.

[0148] In some embodiments, the disease or disorder affects or originates in the central nervous system (CNS). In some embodiments, the disease is a neurodegenerative disease. In some embodiments, the CNS disease or neurodegenerative disease is Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, a polyglutamine repeat disease, or Parkinson's disease. In some embodiments, the CNS disease or neurodegenerative disease is a polyglutamine repeat disease. In some embodiments, the polyglutamine repeat disease is spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0149] In some embodiments, the AAV particles comprise a heterologous nucleic acid encoding a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (IGF), insulin growth factors I and II (IGF-I and IGF-I), TGFp, activins, inhibins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5,ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog genes, and tyrosine hydroxylase.

[0150] In some embodiments, the AAV particles comprise a heterologous nucleic acid encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors a1p, interferons α, β, and γ, stem cell factor, f1k-2 / flt3 ligand, IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, MHC class I and class I1 molecules.

[0151] In some embodiments, the AAV particles comprise a heterologous nucleic acid encoding a gene product selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumaryl acetate hydrolase, phenylalanine hydroxylase, alpha-1-antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H protein, T protein, cystic fibrosis transmembrane receptor (CFTR) sequence, and dystrophin cDNA sequence.

[0152] In some embodiments, the AAV particles comprise a heterologous nucleic acid encoding a polypeptide, a nucleic acid that encodes a protein or is transcribed into a transcript of interest, or a nucleic acid selected from the group consisting of siRNA, antisense molecule, microRNA, ribozyme, and shRNA.

[0153] In some embodiments, the AAV particles comprise a heterologous nucleic acid that encodes a protein selected from the group consisting of GAA (acid alpha-glucosidase) for treating Pompe disease; ATP7B (copper-transporting ATPase2) for treating Wilson's disease; alpha-galactosidase for treating Fabry disease; ASS1 (arginosuccinate synthase) for treating citrullinemia type 1; beta-glucocerebrosidase for treating Gaucher disease type 1; beta-hexosaminidase A for treating Tay-Sachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for treating hereditary angioedema (HAE), also known as C1 inhibitor deficiency type I and type II); and glucose-6-phosphatase for treating glycogen storage disease type I (GSDI).

[0154] In some embodiments, the heterologous nucleic acid encodes CFTR (cystic fibrosis transmembrane regulator protein), a blood coagulation factor (factor XIII, factor IX, factor VIII, factor X, factor VII, factor Vila, protein C, etc.), a factor enhancing coagulation function, an antibody, a 65 kDa protein specific for the retinal pigment epithelium (RPE65), erythropoietin, an LDL receptor, lipoprotein lipase, ornithine transcarbamylase, B-globin, a-globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyltransferase, B-2 5 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, hormone, growth factor, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and -4,brain-derived neurotrophic factor, glial growth factor, transforming growth factor α and β, cytokine, α-interferon, β-interferon, interferon-γ interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance protein, tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), peptide with immunomodulatory properties, tolerogenic or immunogenic peptide or protein Tregitop or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (choroideremia), LCA 5 (LCA-Lebercillin), ornithine ketoacid aminotransferase (circular atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), GTPase X-linked retinitis pigmentosa (XLRP),MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin 2 6 deafness), ACHM 2, 3, and 4 (achromatopsia), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activator protein, one or more zinc finger nucleases for genome editing, or one or more donor sequences used as repair templates for genome editing.

[0155] Nucleic acid molecules, vectors such as cloning vectors, expression vectors (e.g., vector genomes), and plasmids, can be produced using recombinant DNA technology. The availability of nucleotide sequence information allows for the production of nucleic acid molecules in a variety of ways. For example, heterologous nucleic acid encoding factor IX (FIX), containing a vector or plasmid, can be produced using various standard cloning technologies, recombinant DNA, cell expression, or in vitro translation and chemical synthesis methods. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization or computer database screening methods.Such methods include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides sharing common structural features, such as using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of hybridizing to the nucleic acid sequence of interest; (4) computer searches for related sequences in sequence databases; and (5) differential screening of a subtractive nucleic acid library.

[0156] EXAMPLES

[0157] The following examples merely illustrate the subject matter of the invention disclosed herein and should not be construed as limiting in any way.

[0158] Example 1. Rep-degron constructs and rAAV production

[0159] Various constructs were developed to engineer a mechanism for controlled Rep accumulation and increased rAAV production. The Rep transgene was tagged at the N-terminus or C-terminus with a destabilization domain (degron) to confer instability and target the translated Rep protein product to the proteasome for degradation. Rep protein degradation can be reversed by the addition of Shieldl ligand. The degron was modified from the human gene encoding FK506-binding protein 12 (FKBP12). Modifications to the F36V protein have previously been shown to improve its specificity for Shieldl compared to FK506 (Clackson et al, PNAS 1998). Subsequent attempts to engineer a more unstable degron motif resulted in the L107P mutation (Banaszynski et al, Cell 2006). The amino acid sequence of the degron used in this example, herein referred to as "FKBP", having the F36V and L107P mutation, was:

[0160] MGVQVE TIS PGDGRT FPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEV IRGWEE GVAQMSVGQRAKLTIS PDYAYGATGHPGIIPPHATLVFDVELLKPE

[0161] rAAV production. Recombinant AAVs were generated by transfection of human embryonic kidney (HEK293) cells grown in 6-well plates. Cells at 70-90% confluence were transfected with 3 μg of total DNA using jetOPTIMUS® DNA transfection reagent (PolyPlus). Positive control rAAV, generated using the Rep / Cap plasmid LK03, was generated by triple transfection. Plasmids were added at a 1:1:1 ratio (1 helper: 1 ITR plasmid: 1 rep / cap plasmid). rAAV plasmids generated using digested Rep / Cap plasmids were generated by quadruple plasmid transfection, with Rep and Cap plasmids used in equal amounts, but half the helper plasmids and ITR (1 helper : 1 ITR plasmid : 0.5 Rep : 0.5 cap), unless otherwise noted. For degron-containing constructs, WT Rep or Rep / Cap plasmids were replaced with degron-containing Rep or Rep / Cap plasmids. After transfection, the cell culture medium was changed the following day.The small molecule Shield-1 (Takara) was added to the medium of the respective samples after medium replacement, and the cells were grown for an additional 24-36 hours to ensure AAV production. AAV in crude cell lysate extract or in HEK2 93 cell medium was used to transduce target cells.

[0162] To use rAAV in the crude extract, cells were resuspended in the cell culture medium in which they were grown, and the suspension was transferred to 1.5 ml tubes. Instead of lysing with detergents, cells were lysed by four consecutive freeze-thaw cycles, rotating between dry ice and a 37°C water bath. After each thawing cycle, samples were vortexed to enhance cell lysis and release the viral promoter. After completion of the lysis steps, samples were centrifuged at 13,500 x g for 10 minutes in an Eppendorf centrifuge set at 4°C. The supernatant containing rAAV particles was transferred to a fresh tube for use in transduction assays.

[0163] Cell transduction. Huh7 cells were transduced with an equivalent volume of cell lysate or cell medium containing AAV vectors. Briefly, Huh7 cells were seeded in 24-well plates the day before transduction. Up to 50 μl of crude HEK2-93 viral preparations were added to the Huh7 cell culture medium. Transduction efficiency was assessed in two or three biological replicates. The culture medium was replaced the following day, and samples were analyzed for transgene expression 48–72 h after transduction. To detect luciferase expression, Huh7 cells were lysed in passive lysis buffer (Promega), and each biological sample was split and placed into four wells of a 96-well plate for luminescence assay. Renilla luciferase levels were determined using a Renilla luciferase assay kit (Promega) and analyzed on a microplate luminometer (Spectramax) equipped with injectors.

[0164] Cloning of plasmid constructs. Constructs containing degron motifs were cloned using the Gibson assembly method. Briefly, engineered degron-linker sequences with the desired homology regions were ordered as gBlocks fragments from Integrated DNA Technologies. Backbones containing only Rep plasmids or Rep / Cap plasmids and g-blocks were assembled using the NEBuilder Hifi DNA Assembly Kit (New England Biolabs, #E5520). After plasmid purification and sequence verification, plasmids with the correct sequences were used for rAAV production experiments.

[0165] As shown in Figures 1 and 2, the REP and Cap genes were cloned either into the same or separate plasmids. The goal was to regulate Rep expression without altering Cap expression. Plasmids were designed in which degron sequences were cloned either at the C-terminus or N-terminus of the Rep sequence (Figure 1). Two types of linkers were tested: rigid and flexible linkers. The p40 promoter was used to drive Cap expression. As noted, the p5 promoter was used for Rep in some cases, while the CMV promoter was used in others.

[0166] Transfection of cells with the transgene plasmid alone, as shown in Figures 1 and 2, was used as a negative control. Shield1 was added to selected wells (Figure 2). Huh7 cells (liver cells, hepatocellular carcinoma) were transduced using medium or cell lysates from rAAV-producing HEK2 93 cells. Huh7 cells were then lysed, and luciferase activity was measured to detect transgene activity.

[0167] Example 2. rAAV obtained by transfection of Rep and Cap on separate plasmids

[0168] The methods of Example 1, unless otherwise noted, were used to determine whether rAAV could still be produced by transfecting Rep and Cap on separate plasmids. A Rep / Cap plasmid without a degron, a Rep plasmid without a degron, and a Cap plasmid only were tested. Rep was under the control of the CMV promoter. For Cap, the CMV promoter (CMV-Cap (*), which lacks the Rep end) and the P40 promoter (P40-Cap, which contains the Rep end) were used. As shown in Figure 3, P40-Cap could rescue rAAV production, as determined by transduction of target cells. However, CMV-Cap could not rescue rAAV production. Figure 3 shows that rAAV could be produced by transfecting Rep and Cap on separate plasmids.

[0169] Example 3. Regulation of rAAV production by addition of Shield1

[0170] The methods of Example 1, unless otherwise noted, were used to determine whether the addition of Shieldl could posttranslationally control Rep degron accumulation. Constructs were transfected into HEK2 93 cells with or without the Cap plasmid (Figure 4). The addition of Shieldl rescued rAAV production and transduction of target cells. As shown in Figure 4, only the C-terminal degron of the Rep plasmid was used. Transduction efficiency was 15-20% of the Rep / Cap plasmid mass.

[0171] Example 4. Regulation of Different Rep Constructs The methods of Example 1, unless otherwise noted, were used to determine whether the location of the degron (i.e., the degron located at the C-terminus or N-terminus) affects the post-translational regulation of Rep expression. Huh7 cells were transduced with HEK293 cell supernatant as described above. Plasmid P40-Cap was used to drive Cap expression. As shown in Figures 5 and 10, rAAV production from Rep constructs having a degron linked to the N-terminus exhibited less tightly regulated expression compared to Rep constructs having a degron linked to the C-terminus.

[0172] The methods of Example 1, unless otherwise noted, were used to determine the effect of Shieldl on the post-translational regulation of Rep expression for constructs containing both Rep and Cap coding sequences, compared to constructs containing only Rep coding sequences. Plasmid P40-Cap was used to drive Cap expression, with the "Cap plasmid" (as indicated by the "+" sign) being provided. As shown in Figure 6, although there is a moderate increase in rAAV production when Rep with a C-terminally linked degron is expressed in the presence of Cap (either expressed from a single Rep / Cap construct or if Cap is expressed in trans), rAAV production is significantly increased when Rep with a C-terminally linked degron is expressed in the presence of both Cap and Shieldl. Again, Cap can be expressed from a single Rep / Cap construct or if Cap is expressed in trans.

[0173] The methods of Example 1, unless otherwise noted, were used to determine the effect of adding increasing amounts of a Rep-encoding plasmid with a C-terminal degron on rAAV production. As shown in Figure 7, increasing the amount of Rep-encoding plasmid with a C-terminal degron reduced overall rAAV production and resulted in less differential regulation by Shield1.

[0174] Example 5. Regulation of Rep constructs using rigid or flexible linkers

[0175] The methods of Example 1, unless otherwise stated, were used to determine the effect of the type of linker between the C-terminus of Rep and the degron. Two types of linkers were tested; a rigid and a flexible linker. Figure 8 shows the results for: Rep without degron (+ / - Shieldl); Rep with a rigidly linked C-terminal degron (+ / - Shieldl); Rep with a flexibly linked C-terminal degron (+ / - Shieldl); and controls in which neither Rep nor Cap were provided, and a Rep / Cap double plasmid (in which expression is driven by the distal P5 promoter). Figure 9 shows the results for: Rep without degron (+ / - Shieldl); Rep with a rigidly linked N-terminal degron (+ / - Shieldl); Rep with a flexibly linked N-terminal degron (+ / - Shieldl); and controls in which neither Rep nor Cap were provided, and a double Rep / Cap plasmid (in which expression is driven by the distal P5 promoter).In both Figure 8 and Figure 9, the use of a flexible linker is associated with Shieldl-mediated post-translational control of Rep expression and, consequently, rAAV production.

[0176] Example 6. Analysis of Rep expression and AAV production

[0177] Materials and Methods

[0178] Transfection

[0179] Expi2 93 cells were seeded in Expi2 93 medium at 1.4E6 viable cells / mL one day before transfection. Transfection was performed when the viable cell count was 2E6-3E6 cells / mL. PEI (Polyplus) and OptiMEM (Gibco) transfection reagents were used. The PE1:DNA ratio was 2, and 0.6 μg DNA per million cells was used. Triple transfection was performed using Rep Cap RHM4-1, helper plasmids, and Gaussia luciferase. Digested Rep and Cap plasmids were used at a ratio of 1:4 (Rep:Cap). In some cases, digested Rep Cap and TIR1 or CMV-Tet plasmids were used in a 1:3:1 ratio (Rep:Cap:TIR1 or CMV-Tet). 1.5 mM SAHA was added after the addition of DNA-PEI complexes. In some cases, Shieldl, TMP, auxin, and / or doxycycline were added the following day. For samples treated with doxycycline, a second Dox treatment was performed 24 hours after the first treatment.Cells were harvested at 4, 8, and 24 hours after treatment and at 4, 68, or 72 hours after transfection; centrifuged and washed with cold PBS. These cell pellets were stored in a freezer at -80°C. One ml of cells was harvested at 44, 68, or 72 hours after transfection for qPCR.

[0180] Western blot

[0181] Cell pellets were lysed with RIPA buffer (Pierce), including protease and phosphatase inhibitors and EDTA, on a plate for 30 minutes. The lysates were centrifuged at maximum speed for 15 minutes, and the supernatant was transferred to a new Eppendorf tube and used for Western blotting. Protein concentration was measured using a BCA kit (Pierce). 30 μg of protein per lane was loaded for SDS-PAGE gels. These gels were transferred to a PVDF or nitrocellulose membrane. The membranes were blocked with blocking buffer (Li-Cor) for 30 minutes at room temperature. Purified mouse monoclonal antibody to AAV Rep clone 303.9 (ARP, catalog number 03-61069) was diluted 500-fold with either blocking buffer (Li-Cor) or antibody diluent (Li-Cor). Membranes were incubated overnight in a cold room with or without shaking. The following day, membranes were washed three times for 10 minutes with 1x TBST buffer (Invitrogen).Goat anti-mouse Alexa Flour 680 secondary antibody was diluted 10,000-fold in either blocking buffer (Li-Cor) or antibody diluent (Li-Cor). Membranes were incubated with shaking at room temperature for 45 minutes. Each membrane was then washed six times for 10 minutes with 1x TBST buffer. Membranes were scanned using an Odyssey (Li-Cor). Rabbit anti-GAPDH antibodies (Cell Signaling) were diluted 5,000-fold in either blocking buffer (Li-Cor) or antibody diluent (Li-Cor) as a loading control. The primary incubation was carried out for 1 hour with shaking at room temperature. Goat anti-rabbit secondary antibody Alexa Flour 800 was diluted 10,000-fold with either blocking buffer (Li-Cor) or antibody diluent (Li-Cor). qPCR.

[0182] Frozen harvested cells were thawed and sonicated. 300 µl of sonicated cells were treated with benzonase for 1 hour in an incubator at 37°C on a rotator / shaker. This was followed by treatment with DNase I for 15 minutes. The reaction was stopped with stop reagent (0.2% SDS / 5 mM EDTA / 0.2 M NaCl), heated at 95°C for 10 minutes, and centrifuged. Serial dilutions were performed by 10,000-fold or 100,000-fold. Taqman qPCR was performed using specific primers and probes on a Quant Studio Real-Time PCR system.

[0183] B. Expression of FKBP-tagged Rep protein and AAV production

[0184] In this study, we investigated the regulation of AAV production by Rep proteins using a degron derived from FKBP. Figure 11 preliminarily shows the changes in the molecular weight of the Rep protein upon degron labeling. Specifically, Figure 11 describes the potential size changes in Western blot analysis after adding a degron tag to the N-terminus or C-terminus of the Rep protein. Adding a degron to the N-terminus of the Rep protein (N-degron) only changes the molecular weight of the large Rep protein, but the size of the small Rep protein remains unchanged. The molecular mass of small Rep is not affected by the N-terminal tag because the p19 promoter, which drives expression of small Rep, is located within the Rep gene. Small Rep does not have the same N-terminal sequence as large Rep. In contrast, adding a degron to the C-terminus of Rep (C-degron) changes the molecular mass of both small and large Rep proteins because the C-terminus is the same for both.Adding small molecules such as Shieldl or TMP to cell culture medium can inhibit protein degradation, and the band intensity will change accordingly. The example shown in this paper is a degron derived from FKBP.

[0185] In this study, RHM4-1 was used as the AAV capsid according to the “Materials and Methods” presented in Section 6A above. As shown in Figures 12A–12D, “RHM4-1” and “RHM4-lv2” are plasmids that express both degron-free Rep proteins and RHM4-1 Cap. In contrast, “plasmid p40Cap” expresses only the RHM4-1 capsid, while “plasmid pAAV2 Rep” expresses Rep proteins from AAV2. The ITR plasmid used in these studies contains Gaussia luciferase as a transgene.

[0186] Figure 12A shows a Western blot of N-terminal Rep proteins tagged with the FKBP degron. In this study, a plasmid expressing an N-terminal construct containing the Rep degron (N-degron Rep) was used for AAV production. Samples were collected 4, 8, or 24 hours after the addition of Shieldl to the medium. The molecular weight and expression of small Rep (Rep52) are not affected by the presence or absence of the degron, as explained in Figure 11 (samples 1-6 versus samples 7-11). Control samples generated using degron-free Rep are loaded in lanes 7-11. Due to the inherent plasmid construct, expression of large Rep (Rep78) is much lower than that of small Rep in all samples. Expression of large Rep in Rep N-degron samples increased after addition of Shieldl (samples 4-6) compared to untreated controls (samples 1-3).The molecular weight of the large Rep protein is higher for samples with the Rep N-degron (samples 1-6 versus 7-11), but no change in the molecular weight of the small Rep is observed for these samples, as illustrated in Fig. 11.

[0187] Figure 12B shows a Western blot of FKBP-tagged C-terminal Rep proteins. In this study, a plasmid expressing a C-terminal degron containing the Rep construct (Rep C-degron) was used for AAV production, and samples were collected at different time points (4, 8, and 24 h) after Shieldl induction. Expression of small and large Rep is higher in Shieldl (samples 4-6) than in untreated controls (samples 1-3) at the same time points. Small and large Rep molecular weights are higher in the presence of the FKBP C-terminal degron, and a shift is observed compared to samples lacking degrons containing Rep proteins (samples 1-6 versus 7-11).

[0188] Figure 12B shows the AAV titers of the samples in Figures 12A and 12B. AAV titers are increased by adding the Shieldl molecule to the medium used for AAV production. In this study, cells were transfected with plasmids containing the Rep degron, along with other plasmids required for AA production. Addition of the Shieldl molecule increases AAV titers for both the N- and C-terminal degron constructs compared to the untreated control.

[0189] Figure 12D shows the AAV titers observed using Rep and Cap plasmids transfected at different ratios. In this study, degron-tagged Rep proteins are regulated in cells transfected with different ratios of Rep and Cap plasmids. As shown in the results, AAV production can be regulated by degron-tagged Rep proteins, presented as separate plasmids, transfected at different ratios into HEK293 cells, as indicated at the bottom of the figure. Titers were normalized compared to the titer of a control transfection using a degron-free Rep / Cap plasmid. 500 nM Shieldl was added to the samples to inhibit Rep degradation.

[0190] B. Expression of ecDHFR degron-tagged Rep protein and AAV production

[0191] In this study, we investigated the regulation of AAV production by Rep proteins using a degron derived from ecDHFR. The degron derived from ecDHFR has the following amino acid sequence:

[0192] MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPG RKNIILS SQPGT DDRVTWVKSVDEAlAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHID AEVEGDTHFPDYE PDDWE SVFSEFHDADAQNSHS YCFEILERR.

[0193] The degron derived from ecDHFR has the following DNA sequence:

[0194]

[0195] In this study, RHM4-1 was used as the AAV capsid according to the “Materials and Methods” presented in Section 6A above.

[0196] Figure 13A shows a Western blot of the N-terminal Rep protein tagged with the ecDHFR degron. In this study, the N-terminal ecDegron construct containing Rep (N-ecDegron Rep) was used for AAV production, and the Cap gene was provided as a separate plasmid. The expression of small Rep is not affected by the N-terminal ecDegron fusion (samples 1-6), as explained in Figure 11. The expression of large Rep is higher for samples treated with 5 μM TMP after 24 h (sample 3 compared to sample 6). The molecular weight of large Rep is higher because it is tagged with the ecDHFR degron (N-terminus), and a shift is observed for these samples (samples 1-6). As expected, such a shift is not observed for small Rep proteins, since their molecular weight remains unchanged.

[0197] Figure 13B shows a Western blot of C-terminal Rep proteins tagged with the ecDHFR degron. In this study, a C-terminal ecDegron construct containing Rep (C-ecDegron Rep) was used for AAV production, and the Cap gene was presented as a separate plasmid. Expression of small and large Rep was higher at 5 μM TMP (sample 3 compared to sample 6). The molecular weights of small and large Rep were higher with the C-terminal ecDHFR degron (samples 1-6), and shifts were observed for both proteins.

[0198] Figure 13B shows the AAV titers of samples from Figures 13a and 13b. Titer levels of AAV produced using plasmids containing the Rep ecDHFR degron were analyzed. Addition of TMP increased the AAV titer of C-terminal degron constructs, which correlated with the Western blot data on Rep expression.

[0199] G. Expression of Rep protein tagged with auxin degron or ecDHFR and AAV production

[0200] In this study, we investigated the regulation of AAV production by Rep proteins using an auxin-derived degron or an ecDHFR-derived degron. The auxin-derived degron has the following amino acid sequence:

[0201]

[0202] Degron derived from auxin has the following DNA sequence:

[0203]

[0204] In this study, RHM4-1 was used as the AAV capsid according to the “Materials and Methods” presented in Section 6A above.

[0205] Figure 14A shows a Western blot of Rep tagged with auxin-inducible degron or ecDHFR degron. The small and large Rep sizes are larger in the C-terminal auxin-inducible degrons of Rep-containing plasmids (samples 3–6), and shifts are observed for both proteins. Changes in Rep expression for the auxin-inducible degron are difficult to observe due to the presence of a nonspecific band of the same size as the small Rep-tagged degron. This nonspecific band is present even in the untransfected sample (sample 9).

[0206] Expression of small and large Rep is more pronounced when using TMP (samples 7 and 8) for the C-terminally tagged ecDHFR degron. Small and large Rep sizes increase with the C-terminal tagged ecDHFR degron (samples 7 and 8), and shifts are observed.

[0207] Figure 14B shows the AAV titers of the samples in Figure 14A. As shown in Figure 14C, auxin-inducible degron and ecDHFR degron can regulate AAV titer, and auxin-inducible degrons only function when auxin and TIR1 ubiquitin ligase are present in cells along with auxin-dependent degron. Amino acid

[0208] TIR-1 protein sequence is:

[0209]

[0210] Unlike FKBP and ecDHFR-dependent degrons, which prevent protein degradation upon addition of small molecules, auxin-inducible degron causes degradation of the protein to which it is bound. For auxin-inducible degron to be functional, the presence of auxin, TIR1, and the degron is required. No AAV production was detected in the sample, although all components (degron, auxin, and TIR1) of the auxin-mediated degron were present (sample 4). This demonstrates the functionality of this degron in regulating AAV production at the C-terminus of the Rep protein. Reps tagged with the ecDHFR degron could produce AAV in the presence of a TMP molecule (sample 8), which was undetectable without a TMP molecule (sample 7).

[0211] D. Rep protein expression and AAV production in the presence of Shieldl or dTag13

[0212] Figure 15 shows the effect of different doses of Shieldl and dTag13 on AAV production. In this study, Rep constructs with a C-terminal degron labeled with different doses of Shieldl and dTag13 were tested. Different levels of Shieldl were compared to determine the effect on titer. Shieldl was more effective at a concentration of 500 nM in increasing AAV production. dTag13, a molecule that binds to several FKBP-derived domains and causes their subsequent degradation, was also tested. dTag13 did not reduce the basal level of AAV production in this study.

[0213] E. Rep protein expression and AAV production under the control of transcriptional level control and / or degron level control

[0214] Figure 16A shows a Western blot of Rep with a C-terminal degron under the control of the TRE3G promoter. The DNA sequence of the TRE3G promoter is:

[0215]

[0216] In this study, a Tet response element-containing promoter (TRE3G) was cloned upstream of the Rep gene, as shown in Figure 18A. This promoter could be activated in the presence of Tet proteins and doxycycline induction. Rep with a C-terminal degron under the control of the Tet response element-containing promoter was shown to be functional in regulating AAV production. Large Rep was under the control of the Tet promoter, and small Rep expression was driven by the p19 promoter (samples 1-5). Large Rep expression was tightly controlled by this system and could be detected by the addition of doxycycline (samples 3 and 4). Shieldl could enhance small Rep expression (sample 1 versus sample 2) and large Rep expression (sample 3 versus sample 4). A separate plasmid is used to deliver the Tet-On 3G transactivator protein, with expression of the transactivator protein under the control of the ubiquitous CMV promoter (samples 1-4).The amino acid sequence of the transactivator protein is:

[0217] MSRLDKSKVINSALE LLNGVGIEGLTTRKLAQKLGVE QPT LYWHVKNKRALLDALP

[0218] D Li

[0219]

[0220] The DNA sequence of the CMV promoter is:

[0221]

[0222] Figure 16B shows the AAV titers of the samples in Figure 16A. This study demonstrates that AAV production can be regulated by C-terminal degron-tagged Rep under the control of the TRE3G promoter, and that doxycycline induction can increase AAV production by -2.9-fold compared to basal levels. AAV titer levels in these samples are comparable to those of the control samples without degron (samples 6-8).

[0223] Figure 17A shows a Western blot of Rep constructs with a C-terminal degron. In this study, the effect of different levels of doxycycline on AAV production from Rep-degron constructs under the control of the TRE3G promoter was tested. Large Rep is under the control of the TRE3G promoter (samples 1-10). Doxycycline effectively induces expression of large Rep (samples 3-8). Expression of large and small Rep is increased in the presence of Shield1 (samples 2, 4, 6, and 8 compared to samples 1, 3, 5, and 7). Exclusion of the plasmid that expresses the protein-

[0224] Tet-On 3G transactivator, when transfected, results in inactivation of doxycycline induction (sample 9 and sample 10).

[0225] Figure 17B shows the AAV titers of the samples in Figure 17A. This study demonstrates that Rep under the control of TRE3G and degron can produce amounts of AAV comparable to the positive control, the unregulated Rep / Cap plasmid.

[0226] Different levels of doxycycline can increase AAV titer with or without Shield1 molecule, but the basal level is lower in the absence of Shield (sample 1), which is evidence of tighter regulation by the dual system.

[0227] Figure 18A shows the effect of the p5 promoter on Rep protein levels in the TRE3G-Rep-flerpoH system. In this study, plasmids with degron-tagged Rep constructs under the control of the TRE3G promoter were generated with or without the p5 promoter at the 3' end of the Rep gene.

[0228] Figure 18B shows a Western blot of Rep constructs with a C-terminal degron and the TRE3G promoter. In this study, large Rep is under the control of the TRE3G promoter (samples 1-8). Doxycycline effectively induces expression of large Rep (samples 3-4 and 7-8), and large Rep expression is further increased by the addition of Shield1 (sample 4 and sample 8). As shown in the figure, both plasmids (with or without the p5 promoter at the 3' end of the Rep gene) can express large and small repeats.

[0229] Figure 18B shows the AAV titers of the samples from Figure 18B. This study demonstrates that the P5 promoter may not be essential for AAV production during transient transfection using the TRE3G system. As shown in the figure, both plasmids can produce comparable levels of AAV titers. Gaussia luciferase was again used as the gene of interest (GOI).

[0230] Figures 20A-20B depict codon-modified Rep with a degron motif and demonstrate that such constructs can be used for AAV production. The figures also show a second AAV Rep gene, small Rep, beneath a regulatory element and degron domain. The results achieved with these constructs demonstrate that the disclosed method can be adapted to work with multiple identical or different regulatory elements, multiple identical or different degron domains, and modified or unmodified genes encoding AAV proteins.

[0231] Example 7. Degron-tagged helper genes for regulated AAV production

[0232] As shown in this example, degron-tagged helper genes can be used to regulate AAV production. The classical helper plasmid used for AAV production contains the E2A, E4, and VA genes. In this example, the open reading frames of the E2A, E4, and VA genes were cloned into separate plasmids. The selected open reading frames for E2A, E4, and VA are DBP, E4-E34K, and VA2, respectively. The triple transfection method described in Examples 1–6 was modified to allow delivery of the E2A-DBP, E4-E34K, or VA2 genes in separate plasmids instead of a single helper plasmid for use in triple production. A. Materials and Methods

[0233] For instant degron assays using the helper genes, the DNA-binding protein (DBP), E4-E34K, and VA2 genes were selected as open reading frames for the E2A, E4, and VA genes, respectively. These selected open reading frames for the E2A, E4, and VA genes were cloned into separate plasmids under the control of the CMV promoter. The degron derived from FKBP was cloned into the C-terminus of the E2A-DBP gene.

[0234] The triple transfection method was modified to allow delivery of the E2A-DBP, E4-E34K, and VA2 genes on three separate plasmids instead of a single helper plasmid. The same reagents and methods were used for transfection and cell lysis as in the Rep-degron experiments described in this paper. The molar ratio of plasmids used for quintuple transfections (ITR-GOI: Rep / Cap: DBP: E34K: VA2) to individual helper plasmids was 2:2:1:1:1. The molar ratios for plasmids used for triple transfections (ITR-GOI: Rep / Cap: helper) to a single helper plasmid were 2:2:1. Negative control samples, in which AAV production was prevented, were generated by transfecting cells with only the ITR-GOI plasmid (i.e., helper plasmids and Rep / Cap were excluded). For Western blot analysis of DBP in the experimental results, a polyclonal antibody to DBP from CusaBio (CSB-PA365892ZA01HIL) was used.

[0235] Experiment results

[0236] As shown in Figure 19A, the DBP protein expressed by the E2A gene was tagged with a degron motif derived from FKBP. The E2A-DBP-degron-expressing plasmid was co-transfected with other plasmids expressing Rep / Cap, ITR-GOI, E4-E34K, and VA2. After 72 hours, the cells were lysed, and AAV titer levels were analyzed. Cells transfected with the ITR-GOI plasmid alone (i.e., excluding the helper and Rep / Cap plasmids) were used as negative control samples. Without the addition of the Shield1 molecule, AAV production was observed at the same level as the background levels of the negative control qPCR results (solid band on the left and band on the right). After addition of the Shield1 molecule, titer levels increased approximately 3-fold, demonstrating regulation of AAV production by degron-tagged E2A-DBP (striped middle band).

[0237] Figure 19B shows a Western blot highlighting the size shift of the E2A-DBP protein due to the addition of a degron tag. The tagged protein is -12 kDa larger than the unlabeled DBP protein. The two samples on the left represent unlabeled DBP, while the samples on the right represent DBP with a degron.

[0238] Used helper gene sequences E2A-DBP nucleic acid sequence

[0239]

[0240]

[0241] Amino acid sequence of E2A-DBP

[0242] Nucleic acid sequence E4-34K (0RF6)

[0243]

[0244] Amino acid sequence E4-34K (0RF6)

[0245] Nucleic acid sequence of VA2:

[0246]

[0247] Amino acid sequence of VA2:

[0248]

[0249] All publications, patents and other references cited herein are incorporated into the present invention by reference in their entirety.

[0250] --->

[0251] SEQUENCE LISTING

[0252] <110> SPARK THERAPEUTICS, INC.

[0253] <120> Methods of Regulating the Production of Adeno-Associated Virus

[0254] <130> 089504.0105

[0255] <140> PCT / US2022 / 033071

[0256] <141> 2022-06-10

[0257] <150> US 63 / 209,735

[0258] <151> 2021-06-11

[0259] <150> US 63 / 350,849

[0260] <151> 2022-06-09

[0261] <160> 17

[0262] <170> PatentIn, version 3.5

[0263] <210> 1

[0264] <211> 15

[0265] <212> Protein

[0266] <213> Artificial sequence

[0267] <220>

[0268] <223> Synthetic: Flexible linker

[0269] <400> 1

[0270] Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

[0271] 1 5 10 15

[0272] <210> 2

[0273] <211> 46

[0274] <212> Protein

[0275] <213> Artificial sequence

[0276] <220>

[0277] <223> Synthetic: rigid linker

[0278] <400> 2

[0279] Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

[0280] 1 5 10 15

[0281] Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala

[0282] 20 25 30

[0283] Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

[0284] 35 40 45

[0285] <210> 3

[0286] <211> 108

[0287] <212> Protein

[0288] <213> Artificial sequence

[0289] <220>

[0290] <223> Synthetic: amino acid sequence

[0291] degron (FKBP), which has mutations F36V and L107P

[0292] <400> 3

[0293] Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe

[0294] 1 5 10 15

[0295] Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu

[0296] 20 25 30

[0297] Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys

[0298] 35 40 45

[0299] Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

[0300] 50 55 60

[0301] Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp

[0302] 65 70 75 80

[0303] Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala

[0304] 85 90 95

[0305] Thr Leu Val Phe Asp Val Glu Leu Leu Lys Pro Glu

[0306] 100 105

[0307] <210> 4

[0308] <211> 159

[0309] <212> Protein

[0310] <213> Artificial sequence

[0311] <220>

[0312] <223> Synthetic: degron derived from ecDHFR

[0313] <400> 4

[0314] Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Tyr Val Ile Gly Met

[0315] 1 5 10 15

[0316] Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys

[0317] 20 25 30

[0318] Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu

[0319] 35 40 45

[0320] Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser

[0321] 50 55 60

[0322] Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu

[0323] 65 70 75 80

[0324] Ala Ile Ala Ala Cys Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly

[0325] 85 90 95

[0326] Gly Arg Val Ile Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu

[0327] 100 105 110

[0328] Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr

[0329] 115 120 125

[0330] Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp

[0331] 130 135 140

[0332] Ala Gln Asn Ser His Ser Tyr Cys Phe Glu Ile Leu Glu Arg Arg

[0333] 145 150 155

[0334] <210> 5

[0335] <211> 480

[0336] <212> DNA

[0337] <213> Artificial sequence

[0338] <220>

[0339] <223> Synthetic: degron derived from ecDHFR

[0340] <400> 5

[0341] atgatcagtc tgattgcggc gttagcggta gattacgtta tcggcatgga aaacgccatg 60

[0342] ccgtggaacc tgcctgccga tctcgcctgg tttaaacgca acaccttaaa taaacccgtg 120

[0343] attatgggcc gccatacctg ggaatcaatc ggtcgtccgt tgccaggacg caaaaatatt 180

[0344] atcctcagca gtcaaccggg tacggacgat cgcgtaacgt gggtgaagtc ggtggatgaa 240

[0345] gccatcgcgg cgtgtggtga cgtaccagaa atcatggtga ttggcggcgg tcgcgttatt 300

[0346] gaacagttct tgccaaaagc gcaaaaactg tatctgacgc atatcgacgc agaagtggaa 360

[0347] ggcgacaccc attcccgga ttacgagccg gatgactggg aatcggtatt cagcgaattc 420

[0348] cacgatgctg atgcgcagaa ctctcacagc tattgctttg agattctgga gcggcggtaa 480

[0349] <210> 6

[0350] <211> 148

[0351] <212> Protein

[0352] <213> Artificial sequence

[0353] <220>

[0354] <223> Synthetic: degron derived from auxin

[0355] <400> 6

[0356] Met Gly Ser Val Glu Leu Asn Leu Arg Glu Thr Glu Leu Cys Leu Gly

[0357] 1 5 10 15

[0358] Leu Pro Gly Gly Asp Thr Val Ala Pro Val Thr Gly Asn Lys Arg Gly

[0359] 20 25 30

[0360] Phe Ser Glu Thr Val Asp Leu Lys Leu Asn Leu Asn Asn Glu Pro Ala

[0361] 35 40 45

[0362] Asn Lys Glu Gly Ser Thr Thr His Asp Val Val Thr Phe Asp Ser Lys

[0363] 50 55 60

[0364] Glu Lys Ser Ala Cys Pro Lys Asp Pro Ala Lys Pro Pro Ala Lys Ala

[0365] 65 70 75 80

[0366] Gln Val Val Gly Trp Pro Pro Val Arg Ser Tyr Arg Lys Asn Val Met

[0367] 85 90 95

[0368] Val Ser Cys Gln Lys Ser Ser Gly Gly Pro Glu Ala Ala Ala Phe Val

[0369] 100 105 110

[0370] Lys Val Ser Leu Asp Gly Ala Pro Tyr Leu Arg Lys Ile Asp Leu Arg

[0371] 115 120 125

[0372] Leu Tyr Lys Ser Tyr Asp Glu Leu Ser Asn Ala Leu Ser Asn Leu Phe

[0373] 130 135 140

[0374] Ser Ser Phe Thr

[0375] 145

[0376] <210> 7

[0377] <211> 447

[0378] <212> ДНК

[0379] <213> Read more

[0380] <220>

[0381] <223> Suggestions: дегрон, contact us

[0382] <400> 7

[0383] atgggcagtg tcgagctgaa tctgagggag actgagctgt gtcttggtct tcccggtgga 60

[0384] gatacagtgg ctccggtaac cggaaacaag agagggttct cagagacggt tgatctgaag 120

[0385] ctaaatctga ataatgagcc tgcaaacaag gaaggatcta cgactcatga cgtagtgact 180

[0386] tttgattcca aggagaagag tgcttgtcct aaagatccag ccaaacctcc ggccaaggca 240

[0387] caagttgtgg gatggccacc ggtgagatca taccggaaga acgtgatggt ttcctgccaa 300

[0388] aaatcaagcg gtggcccgga ggcggcggcg ttcgtgaagg tatcattaga cggagcaccg 360

[0389] tacttgagga aaatcgattt gaggttatat aaaagctacg atgagctttc taatgctttg 420

[0390] tccaacttat tcagctcttt tacctga 447

[0391] <210> 8

[0392] <211> 594

[0393] <212> Protein

[0394] <213> Artificial sequence

[0395] <220>

[0396] <223> Synthetic: amino acid sequence of the TIR-1 protein

[0397] <400> 8

[0398] Met Gln Lys Arg Ile Ala Leu Ser Phe Pro Glu Glu Val Leu Glu His

[0399] 1 5 10 15

[0400] Val Phe Ser Phe Ile Gln Leu Asp Lys Asp Arg Asn Ser Val Ser Leu

[0401] 20 25 30

[0402] Val Cys Lys Ser Trp Tyr Glu Ile Glu Arg Trp Cys Arg Arg Lys Val

[0403] 35 40 45

[0404] Phe Ile Gly Asn Cys Tyr Ala Val Ser Pro Ala Thr Val Ile Arg Arg

[0405] 50 55 60

[0406] Phe Pro Lys Val Arg Ser Val Glu Leu Lys Gly Lys Pro His Phe Ala

[0407] 65 70 75 80

[0408] Asp Phe Asn Leu Val Pro Asp Gly Trp Gly Gly Tyr Val Tyr Pro Trp

[0409] 85 90 95

[0410] Ile Glu Ala Met Ser Ser Ser Tyr Thr Trp Leu Glu Glu Ile Arg Leu

[0411] 100 105 110

[0412] Lys Arg Met Val Val Thr Asp Asp Cys Leu Glu Leu Ile Ala Lys Ser

[0413] 115 120 125

[0414] Phe Lys Asn Phe Lys Val Leu Val Leu Ser Ser Cys Glu Gly Phe Ser

[0415] 130 135 140

[0416] Thr Asp Gly Leu Ala Ala Ile Ala Ala Thr Cys Arg Asn Leu Lys Glu

[0417] 145 150 155 160

[0418] Leu Asp Leu Arg Glu Ser Asp Val Asp Asp Val Ser Gly His Trp Leu

[0419] 165 170 175

[0420] Ser His Phe Pro Asp Thr Tyr Thr Ser Leu Val Ser Leu Asn Ile Ser

[0421] 180 185 190

[0422] Cys Leu Ala Ser Glu Val Ser Phe Ser Ala Leu Glu Arg Leu Val Thr

[0423] 195 200 205

[0424] Arg Cys Pro Asn Leu Lys Ser Leu Lys Leu Asn Arg Ala Val Pro Leu

[0425] 210 215 220

[0426] Glu Lys Leu Ala Thr Leu Leu Gln Arg Ala Pro Gln Leu Glu Glu Leu

[0427] 225 230 235 240

[0428] Gly Thr Gly Gly Tyr Thr Ala Glu Val Arg Pro Asp Val Tyr Ser Gly

[0429] 245 250 255

[0430] Leu Ser Val Ala Leu Ser Gly Cys Lys Glu Leu Arg Cys Leu Ser Gly

[0431] 260 265 270

[0432] Phe Trp Asp Ala Val Pro Ala Tyr Leu Pro Ala Val Tyr Ser Val Cys

[0433] 275 280 285

[0434] Ser Arg Leu Thr Thr Leu Asn Leu Ser Tyr Ala Thr Val Gln Ser Tyr

[0435] 290 295 300

[0436] Asp Leu Val Lys Leu Leu Cys Gln Cys Pro Lys Leu Gln Arg Leu Trp

[0437] 305 310 315 320

[0438] Val Leu Asp Tyr Ile Glu Asp Ala Gly Leu Glu Val Leu Ala Ser Thr

[0439] 325 330 335

[0440] Cys Lys Asp Leu Arg Glu Leu Arg Val Phe Pro Ser Glu Pro Phe Val

[0441] 340 345 350

[0442] Met Glu Pro Asn Val Ala Leu Thr Glu Gln Gly Leu Val Ser Val Ser

[0443] 355 360 365

[0444] Met Gly Cys Pro Lys Leu Glu Ser Val Leu Tyr Phe Cys Arg Gln Met

[0445] 370 375 380

[0446] Thr Asn Ala Ala Leu Ile Thr Ile Ala Arg Asn Arg Pro Asn Met Thr

[0447] 385 390 395 400

[0448] Arg Phe Arg Leu Cys Ile Ile Glu Pro Lys Ala Pro Asp Tyr Leu Thr

[0449] 405 410 415

[0450] Leu Glu Pro Leu Asp Ile Gly Phe Gly Ala Ile Val Glu His Cys Lys

[0451] 420 425 430

[0452] Asp Leu Arg Arg Leu Ser Leu Ser Gly Leu Leu Thr Asp Lys Val Phe

[0453] 435 440 445

[0454] Glu Tyr Ile Gly Thr Tyr Ala Lys Lys Met Glu Met Leu Ser Val Ala

[0455] 450 455 460

[0456] Phe Ala Gly Asp Ser Asp Leu Gly Met His His Val Leu Ser Gly Cys

[0457] 465 470 475 480

[0458] Asp Ser Leu Arg Lys Leu Glu Ile Arg Asp Cys Pro Phe Gly Asp Lys

[0459] 485 490 495

[0460] Ala Leu Leu Ala Asn Ala Ser Lys Leu Glu Thr Met Arg Ser Leu Trp

[0461] 500 505 510

[0462] Met Ser Ser Cys Ser Val Ser Phe Gly Ala Cys Lys Leu Leu Gly Gln

[0463] 515 520 525

[0464] Lys Met Pro Lys Leu Asn Val Glu Val Ile Asp Glu Arg Gly Ala Pro

[0465] 530 535 540

[0466] Asp Ser Arg Pro Glu Ser Cys Pro Val Glu Arg Val Phe Ile Tyr Arg

[0467] 545 550 555 560

[0468] Thr Val Ala Gly Pro Arg Phe Asp Met Pro Gly Phe Val Trp Asn Met

[0469] 565 570 575

[0470] Asp Gln Asp Ser Thr Met Arg Phe Ser Arg Gln Ile Ile Thr Thr Asn

[0471] 580 585 590

[0472] Gly Leu

[0473] <210> 9

[0474] <211> 386

[0475] <212> DNA

[0476] <213> Artificial sequence

[0477] <220>

[0478] <223> Synthetic: TRE3G promoter DNA sequence

[0479] <400> 9

[0480] gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag 60

[0481] agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttactccc 120

[0482] tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta 180

[0483] cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag 240

[0484] agaacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc tcgtttagtg 300

[0485] ggcgcgccac cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt 360

[0486] tccgtaccac ttcctaccct cgtaaa 386

[0487] <210> 10

[0488] <211> 248

[0489] <212> Protein

[0490] <213> Artificial sequence

[0491] <220>

[0492] <223> Synthetic: Synthetic: amino acid sequence

[0493] transactivator protein

[0494] <400> 10

[0495] Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu

[0496] 1 5 10 15

[0497] Leu Asn Gly Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln

[0498] 20 25 30

[0499] Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys

[0500] 35 40 45

[0501] Arg Ala Leu Leu Asp Ala Leu Pro Ile Glu Met Leu Asp Arg His His

[0502] 50 55 60

[0503] Thr His Ser Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg

[0504] 65 70 75 80

[0505] Asn Asn Ala Lys Ser Tyr Arg Cys Ala Leu Leu Ser His Arg Asp Gly

[0506] 85 90 95

[0507] Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr

[0508] 100 105 110

[0509] Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu

[0510] 115 120 125

[0511] Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys

[0512] 130 135 140

[0513] Val Leu Glu Glu Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr

[0514] 145 150 155 160

[0515] Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Lys Gln Ala Ile Glu Leu

[0516] 165 170 175

[0517] Phe Asp Arg Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu

[0518] 180 185 190

[0519] Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro

[0520] 195 200 205

[0521] Thr Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala

[0522] 210 215 220

[0523] Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp

[0524] 225 230 235 240

[0525] Phe Asp Leu Asp Met Leu Pro Gly

[0526] 245

[0527] <210> 11

[0528] <211> 655

[0529] <212> DNA

[0530] <213> Artificial sequence

[0531] <220>

[0532] <223> Synthetic: CMV promoter DNA sequence

[0533] <400> 11

[0534] cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt atagtaatc 60

[0535] aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 120

[0536] aaatggcccg cctggctgac cgcccaacga ccccgccca ttgacgtcaa taatgacgta 180

[0537] tgttcccata gtaacgccaa tagggacttt cattgacgt caatgggtgg actatttacg 240

[0538] gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 300

[0539] cgtcaatgac ggtaaatggc ccgcctggca tttgcccag tacatgacct tatgggactt 360

[0540] tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 420

[0541] gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 480

[0542] cattgacgtc aatgggagtt tgttttggca caaaatcaa cgggactttc caaaatgtcg 540

[0543] taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 600

[0544] aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaatt 655

[0545] <210> 12

[0546] <211> 1590

[0547] <212> DNA

[0548] <213> Artificial sequence

[0549] <220>

[0550] <223> Synthetic: E2A-DBP nucleic acid sequence

[0551] <400> 12

[0552] atggccagtc gggaagagga gcagcgcgaa accaccccg agcgcggacg cggtgcggcg 60

[0553] cgacgtccac caaccatgga ggacgtgtcg tcccgtcgc cgtcgccgc gcctcccgc 120

[0554] gcgcccccaa aaaagcggct gaggcggcgt ctcgagtccg aggacgaaga agactcgtca 180

[0555] caagatgcgc tggtgccgcg cacacccagc ccgcggccat cgacctcgac ggcggatttg 240

[0556] gccattgcgt ccaaaaagaa aaagaagcgc cctctccca agcccgagcg cccgccatcc 300

[0557] ccagaggtga tcgtggacag cgaggaagaa agagaagatg tggcgctaca aatggtgggt 360

[0558] ttcagcaacc caccggtgct aatcaagcac ggcaagggag gtaagcgcac ggtgcggcgg 420

[0559] ctgaatgaag acgacccagt ggcgcggggt atgcggacgc aagagaaaa ggagagtcc 480

[0560] agtgaagcgg aaagtgaaag cacggtgata aacccgctga gcctgccgat cgtgtctgcg 540

[0561] tgggagaagg gcatggaggc tgcgcgcgcg ttgatggaca agtaccacgt ggataacgat 600

[0562] ctaaaggcaa acttcaagct actgcctgac caatggaag ctctggcggc cgtatgcaag 660 0563

[0564] gtgacgatga tggggcgatt cctgcaggcg tacctgcagt cgtttgcaga ggtaacctac 780

[0565] aagcaccacg agcccacggg ctgcgcgttg tggctgcacc gctgcgctga gatcgaaggc 840 0566

[0567] gtgacgagcg aaaacgggca gcgcgcgctg aaggagcagt ctagcaaggc caagatcgtg 960

[0568] aagaaccggt ggggccgaaa tgtggtgcag atctccaaca ccgacgcaag gtgctgcgtg 1020

[0569] catgacgcgg cctgtccggc caatcagttt tccggcaagt cttgcggcat gttcttctct 1080

[0570] gaaggcgcaa aggctcaggt ggcttttaag cagatcaagg ctttcatgca ggcgctgtat 1140

[0571] cctaacgcccc agaccgggca cggtcacctt ctgatgccac tacggtgcga gtgcaactca 1200

[0572] aagcctgggc atgcaccctt tttgggaagg cagctaccaa agttgactcc gttcgccctg 1260

[0573] agcaacgcgg aggacctgga cgcggatctg atctccgaca agagcgtgct ggccagcgtg 1320

[0574] caccacccgg cgctgatagt gttccagtgc tgcaaccctg tgtatcgcaa ctcgcgcgcg 1380

[0575] cagggcggag gccccaactg cgacttcaag atatcggcgc ccgacctgct aaacgcgttg 1440

[0576] gtgatggtgc gcagcctgtg gagtgaaaac ttcaccgagc tgccgcggat ggttgtgcct 1500

[0577] gagtttaagt ggagcactaa acaccagtat cgcaacgtgt ccctgccagt ggcgcatagc 1560

[0578] gatgcgcggc agaacccctt tgatttttaa 1590

[0579] <210> 13

[0580] <211> 529

[0581] <212> Beloved

[0582] <213> Artificial sequence

[0583] <220>

[0584] <223> Synthetic: amino acid sequence of E2A-DBP

[0585] <400> 13

[0586] Met Ala Ser Arg Glu Glu Glu Gln Arg Glu Thr Thr Pro Glu Arg Gly

[0587] 1 5 10 15

[0588] Arg Gly Ala Ala Arg Arg Pro Pro Thr Met Glu Asp Val Ser Ser Pro

[0589] 20 25 30

[0590] Ser Pro Ser Pro Pro Pro Pro Arg Ala Pro Pro Lys Lys Arg Leu Arg

[0591] 35 40 45

[0592] Arg Arg Leu Glu Ser Glu Asp Glu Glu Asp Ser Ser Gln Asp Ala Leu

[0593] 50 55 60

[0594] Val Pro Arg Thr Pro Ser Pro Arg Pro Ser Thr Ser Thr Ala Asp Leu

[0595] 65 70 75 80

[0596] Ala Ile Ala Ser Lys Lys Lys Lys Lys Arg Pro Ser Pro Lys Pro Glu

[0597] 85 90 95

[0598] Arg Pro Pro Ser Pro Glu Val Ile Val Asp Ser Glu Glu Glu Arg Glu

[0599] 100 105 110

[0600] Asp Val Ala Leu Gln Met Val Gly Phe Ser Asn Pro Pro Val Leu Ile

[0601] 115 120 125

[0602] Lys His Gly Lys Gly Gly Lys Arg Thr Val Arg Arg Leu Asn Glu Asp

[0603] 130 135 140

[0604] Asp Pro Val Ala Arg Gly Met Arg Thr Gln Glu Glu Lys Glu Glu Ser

[0605] 145 150 155 160

[0606] Ser Glu Ala Glu Ser Glu Ser Thr Val Ile Asn Pro Leu Ser Leu Pro

[0607] 165 170 175

[0608] Ile Val Ser Ala Trp Glu Lys Gly Met Glu Ala Ala Arg Ala Leu Met

[0609] 180 185 190

[0610] Asp Lys Tyr His Val Asp Asn Asp Leu Lys Ala Asn Phe Lys Leu Leu

[0611] 195 200 205

[0612] Pro Asp Gln Val Glu Ala Leu Ala Ala Val Cys Lys Thr Trp Leu Asn

[0613] 210 215 220

[0614] Glu Glu His Arg Gly Leu Gln Leu Thr Phe Thr Ser Asn Lys Thr Phe

[0615] 225 230 235 240

[0616] Val Thr Met Met Gly Arg Phe Leu Gln Ala Tyr Leu Gln Ser Phe Ala

[0617] 245 250 255

[0618] Glu Val Thr Tyr Lys His His Glu Pro Thr Gly Cys Ala Leu Trp Leu

[0619] 260 265 270

[0620] His Arg Cys Ala Glu Ile Glu Gly Glu Leu Lys Cys Leu His Gly Ser

[0621] 275 280 285

[0622] Ile Met Ile Asn Lys Glu His Val Ile Glu Met Asp Val Thr Ser Glu

[0623] 290 295 300

[0624] Asn Gly Gln Arg Ala Leu Lys Glu Gln Ser Ser Lys Ala Lys Ile Val

[0625] 305 310 315 320

[0626] Lys Asn Arg Trp Gly Arg Asn Val Val Gln Ile Ser Asn Thr Asp Ala

[0627] 325 330 335

[0628] Arg Cys Cys Val His Asp Ala Ala Cys Pro Ala Asn Gln Phe Ser Gly

[0629] 340 345 350

[0630] Lys Ser Cys Gly Met Phe Phe Ser Glu Gly Ala Lys Ala Gln Val Ala

[0631] 355 360 365

[0632] Phe Lys Gln Ile Lys Ala Phe Met Gln Ala Leu Tyr Pro Asn Ala Gln

[0633] 370 375 380

[0634] Thr Gly His Gly His Leu Leu Met Pro Leu Arg Cys Glu Cys Asn Ser

[0635] 385 390 395 400

[0636] Lys Pro Gly His Ala Pro Phe Leu Gly Arg Gln Leu Pro Lys Leu Thr

[0637] 405 410 415

[0638] Pro Phe Ala Leu Ser Asn Ala Glu Asp Leu Asp Ala Asp Leu Ile Ser

[0639] 420 425 430

[0640] Asp Lys Ser Val Leu Ala Ser Val His His Pro Ala Leu Ile Val Phe

[0641] 435 440 445

[0642] Gln Cys Cys Asn Pro Val Tyr Arg Asn Ser Arg Ala Gln Gly Gly Gly

[0643] 450 455 460

[0644] Pro Asn Cys Asp Phe Lys Ile Ser Ala Pro Asp Leu Leu Asn Ala Leu

[0645] 465 470 475 480

[0646] Val Met Val Arg Ser Leu Trp Ser Glu Asn Phe Thr Glu Leu Pro Arg

[0647] 485 490 495

[0648] Met Val Val Pro Glu Phe Lys Trp Ser Thr Lys His Gln Tyr Arg Asn

[0649] 500 505 510

[0650] Val Ser Leu Pro Val Ala His Ser Asp Ala Arg Gln Asn Pro Phe Asp

[0651] 515 520 525

[0652] Phe

[0653] <210> 14

[0654] <211> 885

[0655] <212> DNA

[0656] <213> Artificial sequence

[0657] <220>

[0658] <223> Synthetic: nucleic acid sequence E4-34K (ORF6)

[0659] <400> 14

[0660] atgactacgt ccggcgttcc atttggcatg acactacgac caacacgatc tcggttgtct 60

[0661] cggcgcactc cgtacagtag ggatcgccta cctccttttg agacagagac ccgcgctacc 120

[0662] atactggagg atcatccgct gctgcccgaa tgtaacactt tgacaatgca caacgtgagt 180

[0663] tacgtgcgag gtcttccctg cagtgtggga tttacgctga ttcaggaatg ggttgttccc 240

[0664] tgggatatgg ttctgacgcg ggaggagctt gtaatcctga ggaagtgtat gcacgtgtgc 300

[0665] ctgtgttgtg ccacattga tatcatgacg agcatgatga tccatggtta cgagtcctgg 360

[0666] gctctccact gtcattgttc cagtcccggt tccctgcagt gcatagccgg cgggcaggtt 420

[0667] ttggccagct ggtttaggat ggtggtggat ggcgccatgt ttaatcagag gtttatatgg 480

[0668] taccgggagg tggtgaatta caacatgcca aaagaggtaa tgtttatgtc cagcgtgttt 540

[0669] atgaggggtc gccacttaat ctacctgcgc ttgtggtatg atggccacgt gggttctgtg 600

[0670] gtccccgcca tgagctttgg atacagcgcc ttgcactgtg ggattttgaa caatattgtg 660

[0671] gtgctgtgct gcagttactg tgctgattta agtgagatca gggtgcgctg ctgtgcccgg 720

[0672] aggacaaggc gtctcatgct gcgggcggtg cgaatcatcg ctgaggagac cactgccatg 780

[0673] ttgtattcct gcaggacgga gcggcggcgg cagcagttta ttcgcgcgct gctgcagcac 840

[0674] caccgcccta tcctgatgca cgattatgac tctaccccca tgtag 885

[0675] <210> 15

[0676] <211> 422

[0677] <212> Protein

[0678] <213> Artificial sequence

[0679] <220>

[0680] <223> Synthetic: amino acid sequence E4-34K (ORF6)

[0681] <400> 15

[0682] Met Thr Thr Ser Gly Val Pro Phe Gly Met Thr Leu Arg Pro Thr Arg

[0683] 1 5 10 15

[0684] Ser Arg Leu Ser Arg Arg Thr Pro Tyr Ser Arg Asp Arg Leu Pro Pro

[0685] 20 25 30

[0686] Phe Glu Thr Glu Thr Arg Ala Thr Ile Leu Glu Asp His Pro Leu Leu

[0687] 35 40 45

[0688] Pro Glu Cys Asn Thr Leu Thr Met His Asn Val Ser Tyr Val Arg Gly

[0689] 50 55 60

[0690] Leu Pro Cys Ser Val Gly Phe Thr Leu Ile Gln Glu Trp Val Val Pro

[0691] 65 70 75 80

[0692] Trp Asp Met Val Leu Thr Arg Glu Glu Leu Val Ile Leu Arg Lys Cys

[0693] 85 90 95

[0694] Met His Val Cys Leu Cys Cys Ala Asn Ile Asp Ile Met Thr Ser Met

[0695] 100 105 110

[0696] Met Ile His Gly Tyr Glu Ser Trp Ala Leu His Cys His Cys Ser Ser

[0697] 115 120 125

[0698] Pro Gly Ser Leu Gln Cys Ile Ala Gly Gly Gln Val Leu Ala Ser Trp

[0699] 130 135 140

[0700] Phe Arg Met Val Val Asp Gly Ala Met Phe Asn Gln Arg Phe Ile Trp

[0701] 145 150 155 160

[0702] Tyr Arg Glu Val Val Asn Tyr Asn Met Pro Lys Glu Val Met Phe Met

[0703] 165 170 175

[0704] Ser Ser Val Phe Met Arg Gly Arg His Leu Ile Tyr Leu Arg Leu Trp

[0705] 180 185 190

[0706] Tyr Asp Gly His Val Gly Ser Val Val Pro Ala Met Ser Phe Gly Tyr

[0707] 195 200 205

[0708] Ser Ala Leu His Cys Gly Ile Leu Asn Asn Ile Val Val Leu Cys Cys

[0709] 210 215 220

[0710] Ser Tyr Cys Ala Asp Leu Ser Glu Ile Arg Val Arg Cys Cys Ala Arg

[0711] 225 230 235 240

[0712] Arg Thr Arg Arg Leu Met Leu Arg Ala Val Arg Ile Ile Ala Glu Glu

[0713] 245 250 255

[0714] Thr Thr Ala Met Leu Tyr Ser Cys Arg Thr Glu Arg Arg Arg Gln Gln

[0715] 260 265 270

[0716] Phe Ile Arg Ala Leu Leu Gln His His Arg Pro Ile Leu Met His Asp

[0717] 275 280 285

[0718] Tyr Asp Ser Thr Pro Met Cys Ile Phe Glu Gln Gly Gly Gly Gly Ser

[0719] 290 295 300

[0720] Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Gly Val Gln Val Glu

[0721] 305 310 315 320

[0722] Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr

[0723] 325 330 335

[0724] Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Val Asp

[0725] 340 345 350

[0726] Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln

[0727] 355 360 365

[0728] Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly

[0729] 370 375 380

[0730] Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr

[0731] 385 390 395 400

[0732] Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val

[0733] 405 410 415

[0734] Glu Leu Leu Lys Pro Glu

[0735] 420

[0736] <210> 16

[0737] <211> 435

[0738] <212> ДНК

[0739] <213> Искусственная последовательность

[0740] <220>

[0741] <223> Synthetic: VA2 nucleic acid sequence

[0742] <400> 16

[0743] atgctgtttc cggaggaatt tgcaagcggg gtcttgcatg acggggaggc aaacccccgt 60

[0744] tcgccgcagt ccggccggcc cgagactcga accgggggtc ctgcgactca acccttggaa 120

[0745] aataaccctc cggctacagg gagcgagcca cttaatgctt tcgctttcca gcctaaccgc 180

[0746] ttacgccgcg cgcggccagt ggccaaaaaa gctagcgcag cagccgccgc gcctggaagg 240

[0747] aagccaaaag gagcgctccc ccgttgtctg acgtcgcaca cctgggttcg acacgcgggc 300

[0748] ggtaaccgca tggatcacgg cggacggccg gatccggggt tcgaaccccg gtcgtccgcc 360

[0749] atgataccct tgcgaattta tccaccagac cacggaagag tgcccgctta caggctctcc 420

[0750] ttttgcacgg tctag 435

[0751] <210> 17

[0752] <211> 144

[0753] <212> Protein

[0754] <213> Artificial sequence

[0755] <220>

[0756] <223> Synthetic: amino acid sequence of VA2

[0757] <400> 17

[0758] Met Leu Phe Pro Glu Glu Phe Ala Ser Gly Val Leu His Asp Gly Glu

[0759] 1 5 10 15

[0760] Ala Asn Pro Arg Ser Pro Gln Ser Gly Arg Pro Glu Thr Arg Thr Gly

[0761] 20 25 30

[0762] Gly Pro Ala Thr Gln Pro Leu Glu Asn Asn Pro Pro Ala Thr Gly Ser

[0763] 35 40 45

[0764] Glu Pro Leu Asn Ala Phe Ala Phe Gln Pro Asn Arg Leu Arg Arg Ala

[0765] 50 55 60

[0766] Arg Pro Val Ala Lys Lys Ala Ser Ala Ala Ala Ala Ala Pro Gly Arg

[0767] 65 70 75 80

[0768] Lys Pro Lys Gly Ala Leu Pro Arg Cys Leu Thr Ser His Thr Trp Val

[0769] 85 90 95

[0770] Arg His Ala Gly Gly Asn Arg Met Asp His Gly Gly Arg Pro Asp Pro

[0771] 100 105 110

[0772] Gly Phe Glu Pro Arg Ser Ser Ala Met Ile Pro Leu Arg Ile Tyr Pro

[0773] 115 120 125

[0774] Pro Asp His Gly Arg Val Pro Ala Tyr Arg Leu Ser Phe Cys Thr Val

[0775] 130 135 140

[0776] <---

Claims

1. A method for regulating the production of recombinant adeno-associated virus (rAAV) vector particles, the method comprising: introduction into the cell: rAAV containing the gene of interest; and a nucleic acid encoding a fusion protein, wherein the fusion protein comprises an AAV protein and a degradation domain dependent on a degradation ligand, culturing the cell under conditions suitable for the production of rAAV vector particles; and bringing the cell into contact with a degradation ligand, whereby the degradation ligand binds to the degradation domain, regulating AAV protein expression and thereby regulating the production of rAAV vector particles.

2. The method of claim 1, wherein the nucleic acid encodes a fusion protein, wherein the fusion protein comprises an AAV protein, a linker, and a degradation domain dependent on a degradation ligand.

3. The method according to claim 1 or 2, wherein the AAV protein is Cap.

4. The method according to claim 1 or 2, wherein the AAV protein is a helper protein.

5. The method according to claim 4, wherein the helper protein is E2.

6. The method according to claim 1 or 2, wherein the AAV protein is Rep.

7. The method according to claims 1, 2, wherein the degradation domain dependent on the degradation ligand is obtained from FKBP.

8. The method according to claims 1-6, wherein the degradation ligand-dependent degradation domain is dihydrofolate reductase (DHFR).

9. The method according to claims 1-6, wherein the degradation ligand-dependent degradation domain is an auxin-inducible degradation domain.

10. The method according to any one of claims 1 to 9, wherein the degradation ligand is a small molecule ligand.

11. The method of claim 7, wherein the small molecule is Shield1.

12. The method of claim 8, wherein the small molecule is trimethoprim (TMP).

13. The method of claim 9, wherein the small molecule is auxin.

14. The method according to any one of claims 1-13, wherein the cell is a cell expressing E1a.

15. The method of claim 14, wherein the cell expressing E1a is a HEK293 cell.

16. The method according to any one of claims 6-15, wherein the Rep protein is a Rep78, Rep68, Rep52 or Rep40 protein.

17. The method according to any one of claims 6-16, wherein the degradation ligand-dependent degradation domain is fused to the C-terminal end of the Rep protein.

18. The method according to any one of claims 6-16, wherein the degradation ligand-dependent degradation domain is fused to the N-terminal end of the Rep protein.

19. The method of claim 2, wherein the linker is a flexible linker.

20. The method of claim 2, wherein the linker is a rigid linker.

21. The method according to any one of claims 1-20, further comprising introducing into the cell a second nucleic acid encoding the Cap protein.

22. The method of claim 21, wherein the nucleic acid encoding the fusion protein and the second nucleic acid encoding the Cap protein are introduced into the cell using at least one plasmid.

23. The method of claim 21, wherein the nucleic acid encoding the fusion protein and the second nucleic acid encoding the Cap protein are introduced into the cell using the same plasmid.

24. The method of claim 21, wherein the nucleic acid encoding the fusion protein and the second nucleic acid encoding the Cap protein are introduced into the cell using separate plasmids.

25. The method according to any one of claims 1-24, wherein the gene encoding the rep protein, the gene encoding the cap protein, or the helper genes are under the control of a regulatory element.

26. The method of claim 25, wherein said regulatory element is a promoter.

27. The method of claim 25, wherein said regulatory element comprises a Tet response binding element.

28. The method according to any one of claims 1-27, wherein the cell is a eukaryotic cell.

29. The method of claim 28, wherein the eukaryotic cell is an animal cell.

30. The method of claim 29, wherein the animal cell is a mammalian cell.

31. The method of claim 30, wherein the mammalian cell is a HEK cell.

32. The method of claim 30, wherein the mammalian cell is a Chinese hamster ovary cell.

33. A cell producing rAAV, wherein the cell comprises a nucleic acid encoding a fusion protein comprising an AAV protein and a degradation ligand-dependent degradation domain.

34. The rAAV-producing cell of claim 33, wherein the nucleic acid encodes a fusion protein comprising an AAV protein, a linker, and a degradation ligand-dependent degradation domain.

35. A cell producing rAAV according to claim 33 or claim 34, wherein the AAV protein is selected from the group consisting of Rep, Cap and helper proteins.

36. The rAAV-producing cell of claim 35, wherein the AAV protein is Cap.

37. The rAAV-producing cell of claim 35, wherein the AAV protein is a helper protein.

38. The rAAV-producing cell of claim 35, wherein the AAV protein is Rep.

39. The rAAV-producing cell of claims 33-38, wherein the degradation ligand is a small molecule ligand.

40. The rAAV-producing cell of claims 33-39, wherein the degradation ligand-dependent degradation domain is derived from FKBP.

41. The rAAV-producing cell of claims 33-39, wherein the degradation ligand-dependent degradation domain is dihydrofolate reductase (DHFR).

42. The rAAV-producing cell of claims 33-39, wherein the degradation ligand-dependent degradation domain is an auxin-inducible degradation domain.

43. The rAAV-producing cell of claim 40, wherein the small molecule is Shield1.

44. The rAAV-producing cell of claim 41, wherein the small molecule is trimethoprim (TMP).

45. The rAAV-producing cell of claim 42, wherein the small molecule is auxin.

46. ​​A rAAV-producing cell according to any one of claims 33-45, wherein the cell is a eukaryotic cell.

47. The rAAV-producing cell of claim 46, wherein the eukaryotic cell is an animal cell.

48. The rAAV-producing cell of claim 47, wherein the animal cell is a mammalian cell.

49. The rAAV-producing cell of claim 48, wherein the mammalian cell is an HEK cell.

50. The rAAV-producing cell of claim 48, wherein the mammalian cell is a Chinese hamster ovary cell.

51. A rAAV-producing cell according to claims 33-45, wherein the cell is an E1a-expressing cell.

52. The rAAV-producing cell of claim 51, wherein the E1a-expressing cell is a HEK293 cell.

53. The rAAV-producing cell of any one of claims 38-45, wherein the ligand-dependent degradation domain is fused via a linker to the C-terminal end of the Rep protein.

54. A cell producing rAAV according to any one of claims 38-45, wherein the cell is a mammalian cell and the ligand-dependent degradation domain is fused via a linker to the N-terminal end of the Rep protein.

55. The rAAV-producing cell of claim 34, wherein the cell is a mammalian cell and the linker is a flexible linker.

56. The rAAV-producing cell of claim 34, wherein the cell is a mammalian cell and the linker is a rigid linker.

57. The rAAV-producing cell of claims 48-56, further comprising introducing into the cell a nucleic acid encoding the Cap protein.

58. The rAAV-producing cell of claim 57, wherein the cell is a mammalian cell and the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using at least one plasmid.

59. The rAAV-producing cell of claim 57, wherein the cell is a mammalian cell and the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using the same plasmid.

60. The rAAV-producing cell of claim 57, wherein the cell is a mammalian cell and the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using separate plasmids.

61. A cell producing rAAV according to any one of claims 48-60, wherein the cell is a mammalian cell and the rep, cap or helper genes are under the control of a regulatory element.

62. The rAAV-producing cell of claim 61, wherein said regulatory element is a promoter.

63. The rAAV-producing cell of claim 61, wherein said regulatory element comprises a Tet response binding element.

64. The method of any one of claims 1-32 or the rAAV-producing cell of any one of claims 33-63, wherein two or more different AAV proteins are independently fused to degradation domains dependent on degradation ligands.

65. The method or cell producing rAAV of claim 64, wherein each individual AAV protein is independently fused to a separate degradation ligand-dependent degradation domain.

Images