Viral vector production systems, engineered cells for viral vector production, and methods of use thereof

Abstract

Described herein are viral vector production systems and engineered cells for viral vector production. Also described herein are methods for producing viral vectors using the engineered cells.

Description

[Technical field]

[0001] Field Described herein are viral vector production systems and engineered cells for viral vector production. Also described herein are methods for producing viral vectors using the engineered cells.

[0002] Related Applications This application claims the benefit under 35 USC § 119 of U.S. Provisional Application No. 63 / 189,771, filed May 18, 2021, the entire contents of which are incorporated herein by reference.

[0003] Reference to a sequence listing submitted as a text file via EFS-WEB This application contains a Sequence Listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on May 17, 2022, is named A121070007WO00-SEQ-CRP and is 15,679 bytes in size. [Background technology]

[0004] background Viral vectors are promising therapeutics that deliver genetic payloads to target cells to treat disease. A common type of payload is DNA encoding a fully functional gene of interest (GOI) to correct defects or mutations in the corresponding gene in the patient's cells. AAV vectors are produced using producer cell lines (e.g., HEK293-derived cell lines) that can produce large amounts of virus, but their growth and production rates can be affected by many factors that affect cell health and resource allocation. Summary of the Invention

[0005] overview Described herein are viral vector production systems and engineered cells for viral vector production that allow for increased control over the expression of payload molecules. Also described herein are methods for using the systems and engineered cells.

[0006] In some aspects, the disclosure relates to a viral vector production system comprising: (a) an engineered cell comprising viral vector production components that comprise one or more heterologous polynucleic acids that collectively encode gene products of a viral vector; (b) a heterologous nucleic acid sequence encoding a first expression cassette, the first expression cassette comprising a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a regulatory RNA; and (c) an imported polynucleic acid comprising a central nucleic acid sequence flanked at the 5' and 3' ends by nucleic acid sequences of viral terminal repeats.

[0007] In some embodiments, the central nucleic acid sequence of the imported polynucleic acid comprises a nucleic acid sequence encoding a multiple cloning site. In some embodiments, the central nucleic acid sequence comprises a multiple cloning sequence flanked by target nucleic acid sequences that are complementary to the regulatory RNA encoded by the first expression cassette. In some embodiments, the multiple cloning sequence is flanked by tandem repeats of target nucleic acid sequences that are complementary to the regulatory RNA encoded by the first expression cassette. In some embodiments, the multiple cloning sequence is flanked at the 5' and 3' ends by target nucleic acid sequences that are complementary to the regulatory RNA encoded by the first expression cassette. In some embodiments, the central nucleic acid sequence further comprises a promoter.

[0008] In some embodiments, the central nucleic acid sequence of the import polynucleic acid sequence comprises a second expression cassette, the second expression cassette comprising a nucleic acid sequence encoding a payload molecule operably linked to both a promoter and a target nucleic acid sequence that is complementary to a regulatory RNA encoded by the first expression cassette.

[0009] In some embodiments, the nucleic acid sequence of the payload molecule comprises a 5'UTR comprising a target nucleic acid sequence that is complementary to a regulatory RNA encoded by the first expression cassette; a 3'UTR comprising a target nucleic acid sequence that is complementary to a regulatory RNA encoded by the first expression cassette; or a combination thereof.

[0010] In some embodiments, the nucleic acid sequence of the payload molecule comprises a tandem repeat of a target nucleic acid sequence that is complementary to a regulatory RNA encoded by the first expression cassette. In some embodiments, the first expression cassette comprises a tandem repeat of a nucleic acid sequence that encodes a regulatory RNA. In some embodiments, the first expression cassette comprises nucleic acid sequences of two or more different regulatory RNAs. In some embodiments, the first expression cassette further comprises a nucleic acid sequence that encodes a gene product of a viral vector production component.

[0011] In some embodiments, the nucleic acid sequence encoding a gene product in the first expression cassette has a 5'UTR that comprises a nucleic acid sequence encoding a regulatory RNA; an intron that comprises a nucleic acid sequence encoding a regulatory RNA; a 3'UTR that comprises a nucleic acid sequence encoding a regulatory RNA; or a combination thereof.

[0012] In some embodiments, the first expression cassette further comprises a nucleic acid sequence encoding a selectable marker. In some embodiments, the selectable marker comprises a fluorescent protein or an antibiotic resistance protein. In some embodiments, the nucleic acid sequence encoding the selectable marker in the first expression cassette has a 5'UTR comprising a nucleic acid sequence encoding a regulatory RNA; an intron comprising a nucleic acid sequence encoding a regulatory RNA; a 3'UTR comprising a nucleic acid sequence encoding a regulatory RNA; or a combination thereof.

[0013] In some embodiments, the viral vector production system is an AAV viral vector production system, and the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of an AAV vector. In some embodiments, the viral vector components comprise the nucleic acid sequences of Rep52 or Rep40; Rep78 or Rep68; E2A; E4Orf6; VARNA; VP1; VP2; VP3; and AAP. In some embodiments, the viral terminal repeat of the imported polynucleic acid is an AAV inverted tandem repeat.

[0014] In some embodiments, the viral vector production system is a lentiviral vector production system, and the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of the lentiviral vector.In some embodiments, the viral vector components comprise the nucleic acid sequences of VSV-G, Gag-Pol and Rev.In some embodiments, the viral terminal repeat of the imported polynucleic acid is a lentiviral long terminal repeat.

[0015] In some embodiments, at least one of the one or more heterologous polynucleic acids of the viral vector production components is stably integrated into the genome of the engineered cell. In some embodiments, each of the one or more heterologous polynucleic acids of the viral vector production components is stably integrated into the genome of the engineered cell. In some embodiments, the engineered cell further comprises a heterologous nucleic acid sequence encoding a first expression cassette.

[0016] In some embodiments, the engineered cells are derived from HEK293 cells, HeLa cells, BHK cells, or Sf9 cells.

[0017] In some embodiments, the regulatory RNA of any one of the above viral vector production systems is an shRNA or amiRNA. In some embodiments, the nucleic acid sequence encoding the shRNA comprises any one of the nucleic acid sequences of SEQ ID NOs: 2-11. In some embodiments, the first expression cassette comprises a nucleic acid sequence encoding a selectable marker, the nucleic acid sequence encoding the selectable marker comprising, 5' to 3', an intron having: (i) an intron donor site; (ii) a nucleic acid sequence encoding the shRNA or amiRNA; and (iii) an intron acceptor site. In some embodiments, the intron comprises tandem repeats of the nucleic acid sequence encoding the shRNA or amiRNA, an shRNA cluster or an amiRNA cluster.

[0018] In some embodiments, the nucleic acid sequence encoding the selectable marker comprises a 5'UTR, where the intron of the selectable marker is located in the 5'UTR; a 3'UTR, where the intron of the selectable marker is located in the 3'UTR; or a combination thereof. In some embodiments, the intron of the selectable marker is located in the coding region of the nucleic acid sequence encoding the selectable marker. In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 12.

[0019] In some aspects, the regulatory RNA of the viral vector production system is a Cas13 guide RNA. In some embodiments, the first expression cassette comprises a constitutive promoter operably linked to a nucleic acid sequence encoding two or more Cas13 guide RNAs. In some embodiments, the viral vector production system further comprises a heterologous polynucleic acid encoding Cas13. In some embodiments, Cas13 is Cas13d. In some embodiments, Cas13d comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:1.

[0020] In some embodiments, the heterologous polynucleic acid encoding Cas13 further comprises a first expression cassette. In some embodiments, the first expression cassette comprises a nucleic acid sequence of a constitutive promoter operably linked to both a nucleic acid sequence encoding a Cas13 gRNA and a nucleic acid sequence encoding Cas13. In some embodiments, the engineered cell further comprises a heterologous polynucleic acid encoding Cas13.

[0021] In some aspects, engineered cells for viral vector production include: (a) viral vector production components that collectively encode the viral vector gene product; (b) a first expression cassette comprising a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding an shRNA or amiRNA; and (c) a transfer polynucleic acid comprising a central nucleic acid sequence flanked at the 5' and 3' ends by nucleic acid sequences of viral terminal repeats, the central nucleic acid sequence comprising a nucleic acid sequence encoding a payload molecule operably linked to both a promoter and a target nucleic acid sequence complementary to the shRNA or amiRNA encoded by the first expression cassette. The heterologous polynucleic acids comprise one or more heterologous polynucleic acids collectively comprising:

[0022] In some embodiments, the nucleic acid sequence of the payload molecule comprises a 5'UTR comprising a target nucleic acid sequence complementary to the shRNA or amiRNA encoded by the first expression cassette; a 3'UTR comprising a target nucleic acid sequence complementary to the shRNA or amiRNA encoded by the first expression cassette; or a combination thereof. In some embodiments, the nucleic acid sequence of the payload molecule comprises tandem repeats of a target nucleic acid sequence complementary to the shRNA or amiRNA encoded by the first expression cassette.

[0023] In some embodiments, the first expression cassette comprises tandem repeats of a nucleic acid sequence encoding an shRNA or amiRNA, an shRNA cluster or an amiRNA cluster. In some embodiments, the first expression cassette comprises nucleic acid sequences of two or more different shRNAs or two or more different amiRNAs. In some embodiments, the first expression cassette comprises a nucleic acid sequence encoding a selectable marker, the nucleic acid sequence encoding the selectable marker comprising, 5' to 3': (i) an intron donor site; (ii) a nucleic acid sequence encoding an shRNA or amiRNA; and (iii) an intron acceptor site.

[0024] In some embodiments, the intron comprises a tandem repeat of a nucleic acid sequence encoding an shRNA or amiRNA, an shRNA cluster or an amiRNA cluster. In some embodiments, the nucleic acid sequence encoding a selectable marker comprises a 5'UTR, where the intron of the selectable marker is located in the 5'UTR; a 3'UTR, where the intron of the selectable marker is located in the 3'UTR; or a combination thereof.

[0025] In some embodiments, the selectable marker intron is located in the coding region of the nucleic acid sequence encoding the selectable marker.

[0026] In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO:12.

[0027] In some embodiments, the nucleic acid sequence encoding the shRNA comprises any of the nucleic acid sequences of SEQ ID NOs: 2-11.

[0028] In some embodiments, the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of an AAV vector. In some embodiments, the viral vector components comprise the nucleic acid sequences of Rep52 or Rep40; Rep78 or Rep68; E2A; E4Orf6; VARNA; VP1; VP2; VP3; and AAP. In some embodiments, the viral terminal repeat of the imported polynucleic acid is an AAV inverted tandem repeat.

[0029] In some embodiments, the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of lentiviral vector.In some embodiments, the viral vector components comprise the nucleic acid sequences of VSV-G, Gag-Pol and Rev.In some embodiments, the viral terminal repeat of the imported polynucleic acid is a lentiviral long terminal repeat.

[0030] In some embodiments, at least one of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell. In some embodiments, each of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell.

[0031] In some embodiments, the engineered cells are derived from HEK293 cells, HeLa cells, BHK cells, or Sf9 cells.

[0032] In some aspects, the present application discloses a method of reducing expression of a payload molecule during viral vector production in any one of the engineered cells described above, comprising expressing an shRNA during viral vector production.

[0033] In some aspects, the application discloses engineered cells for viral vector production comprising one or more heterologous polynucleic acids that collectively comprise: (a) viral vector production components that collectively encode gene products of the viral vector; (b) a first expression cassette comprising a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a Cas13 guide RNA; (c) a nucleic acid sequence encoding Cas13; and (d) a transfer polynucleic acid comprising a central nucleic acid sequence flanked at 5' and 3' ends by nucleic acid sequences of viral terminal repeats, the central nucleic acid sequence comprising a nucleic acid sequence encoding a payload molecule operably linked to both a promoter and a target nucleic acid sequence complementary to the Cas13 guide RNA encoded by the first expression cassette.

[0034] In some embodiments, the first expression cassette comprises a constitutive promoter operably linked to a nucleic acid sequence encoding two or more Cas13 guide RNAs. In some embodiments, the Cas13 is Cas13d. In some embodiments, the Cas13d comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first expression cassette further comprises a nucleic acid sequence encoding Cas13.

[0035] In some embodiments, the first expression cassette comprises a nucleic acid sequence of a constitutive promoter operably linked to both a nucleic acid sequence encoding a Cas13 gRNA and a nucleic acid sequence encoding Cas13. In some embodiments, the nucleic acid sequence of the payload molecule comprises a 5'UTR comprising a target nucleic acid sequence complementary to the Cas13 guide RNA encoded by the first expression cassette; a 3'UTR comprising a target nucleic acid sequence complementary to the Cas13 guide RNA encoded by the first expression cassette; or a combination thereof.

[0036] In some embodiments, the nucleic acid sequence of the payload molecule comprises a tandem repeat of a target nucleic acid sequence that is complementary to the Cas13 guide RNA encoded by the first expression cassette. In some embodiments, the first expression cassette comprises a tandem repeat of a nucleic acid sequence that encodes the Cas13 guide RNA.

[0037] In some embodiments, the first expression cassette comprises nucleic acid sequences of two or more different Cas13 guide RNAs.

[0038] In some embodiments, the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of an AAV vector. In some embodiments, the viral vector components comprise the nucleic acid sequences of Rep52 or Rep40; Rep78 or Rep68; E2A; E4Orf6; VARNA; VP1; VP2; VP3; and AAP. In some embodiments, the viral terminal repeat of the imported polynucleic acid is an AAV inverted tandem repeat.

[0039] In some embodiments, the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of lentiviral vector.In some embodiments, the viral vector components comprise the nucleic acid sequences of VSV-G, Gag-Pol and Rev.In some embodiments, the viral terminal repeat of the imported polynucleic acid is a lentiviral long terminal repeat.

[0040] In some embodiments, at least one of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell. In some embodiments, each of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell.

[0041] In some embodiments, the engineered cells are derived from HEK293 cells, HeLa cells, BHK cells, or Sf9 cells.

[0042] In some aspects, the disclosure relates to a method of reducing expression of a payload molecule during viral vector production in any one of the engineered cells described herein, comprising expressing Cas13 and Cas13 guide RNA during viral vector production.

[0043] The following drawings form part of this specification and are included to further demonstrate certain aspects of the disclosure that may be better understood by reference to one or more of these drawings in combination with the detailed description of certain embodiments presented herein. It should be understood that the data presented in the drawings are not intended to limit the scope of the disclosure in any way. [Brief description of the drawings]

[0044] [Figure 1] Figure 1 shows an exemplary schematic of shRNA-mediated knockdown of an AAV payload molecule (or gene of interest, "GOI"). 1) An RNA is transcribed corresponding to the GOI, a gene with an shRNA in its 3'UTR (here Neo-TagBFP). The shRNA (loop) is flanked by intronic donor and acceptor sites (asterisks). The GOI is flanked by tandem repeats of shRNA target sites in the 5' and 3'UTR (shaded boxes). 2) The shRNA sequence is spliced ​​out, releasing the shRNA hairpin. 3) The shRNA hairpin is processed by the RNAi machinery, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). 4) The RISC with the guide strand binds to the GOI mRNA. 5) The RISC can cleave the GOI mRNA and / or suppress its translation. The reduction in GOI mRNA available for translation results in reduced levels of the GOI protein. [Figure 2A-B]Figure 2A-Figure 2B show a comparison of payload molecule (EGFP) knockdown in cells with or lacking an shRNA expression plasmid. Figure 2A shows fluorescent images of AAV producing cells before harvesting AAV and AAV infection titers with or without the addition of EGFP shRNA. Figure 2B shows quantification of AAV titers. [Diagram 3] Figure 3 shows a comparison of immunoglobulin (IG)-EYFP payload molecules containing an FF5 target site in either the 5'UTR or 3'UTR, an IG-EYFP payload molecule lacking an FF5 target site, and an EGFP payload molecule lacking an FF5 target site. On the left are the fluorescence geometric means of EYFP or EGFP measured in transfected cells that have been used to produce AAV. On the right are the viral titers measured using a transduction assay. Transfection with FF5 shRNA (blue bars) results in a significant reduction of payload molecules in AAV-producing cells for constructs with an FF5 target site, but still results in similar or better viral titers compared to controls not transfected with FF5 shRNA (red bars). [Figure 4] Figure 4 shows an exemplary schematic of Cas13-mediated knockdown of an AAV payload molecule (or gene of interest, "GOI"). The knockdown system consists of a series of target sequences located in the 5'UTR, 3'UTR or both regions that are recognized by the Cas13d-crRNA complex. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Detailed Description The inventors of the present disclosure have recognized that expression of payload molecules (i.e., gene of interest or "GOI") of viral vectors during viral vector production may redirect resources away from viral vector production and toward payload RNA and protein production. The inventors have also recognized that some payload molecules may be toxic and may further affect the growth and productivity of producer cells. Described herein are viral vector production systems and engineered cells for viral vector production that allow for increased control over the expression of payload molecules during viral vector production. Methods of using the engineered cells are also described herein.

[0046] I. Viral Vector Production System In some aspects, the present disclosure relates to a viral vector production system.The viral vector production system described herein comprises: (a) viral vector production components; (b) a first expression cassette, comprising the nucleic acid sequence of a promoter operably linked to the nucleic acid sequence encoding regulatory RNA; and (c) one or more polynucleic acids, collectively comprising imported polynucleic acid.

[0047] As used herein, the term "viral vector production components" refers to one or more polynucleic acids that collectively code the gene products required for the production of viral vectors in recombinant host cells.Several types of viral vectors (including the components required for their production) have been previously described, including adenoviral vectors, adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, and herpes simplex virus vectors.The viral vector production system described herein may include viral vector production components that code the gene products required for the production of any of these aforementioned viral vectors.

[0048] In some embodiments, the viral vector production components include one or more polynucleotides that collectively encode gene products necessary for generating AAV vectors in recombinant host cells. Exemplary AAV gene products include Rep52, Rep40, Rep78, Rep68, E2A, E4Orf6, VARNA, VP1, VP2, VP3, AAP, and MAAP or functional variants thereof. Rep gene products (including Rep52, Rep40, Rep78, and Rep68) are involved in AAV genome replication. E2A gene products are involved in supporting DNA synthesis processivity during AAV replication. E4Orf6 gene products support AAV replication. VARNA gene products play a role in regulating translation. CAP gene products (including VP1, VP2, VP3) encode viral capsid proteins. AAP gene products play a role in capsid assembly. MAAP is a frameshifted VP1 protein that appears to play a role in viral capsid, as described in Ogden et al. Science 366.6469(2019):1139-1143, which is incorporated by reference in its entirety.

[0049] As used herein, the term "functional variant" refers to a gene product that comprises a modified nucleic acid or amino acid sequence compared to a wild-type sequence and can perform the function (e.g., enzymatic, regulatory, or binding) of the wild-type gene product.For example, the functional variant of Rep52 can still function in AAV genome replication.

[0050] In some embodiments, the viral vector components include one or more polynucleotides that collectively encode the gene products: Rep52 (or a functional variant thereof) or Rep40 (or a functional variant thereof); Rep78 (or a functional variant thereof) or Rep68 (or a functional variant thereof); E2A (or a functional variant thereof); E4Orf6 (or a functional variant thereof); VARNA (or a functional variant thereof); VP1 (or a functional variant thereof); VP2 (or a functional variant thereof); VP3 (or a functional variant thereof); and AAP (or a functional variant thereof). In some embodiments, the viral vector components include one or more polynucleotides that collectively encode the gene products: Rep52 (or a functional variant thereof), Rep40 (or a functional variant thereof), Rep78 (or a functional variant thereof), Rep68 (or a functional variant thereof), E2A (or a functional variant thereof), E4Orf6 (or a functional variant thereof (e.g., SEQ ID NO: 23), VARNA (or a functional variant thereof), VP1 (or a functional variant thereof), VP2 (or a functional variant thereof), VP3 (or a functional variant thereof), and AAP (or a functional variant thereof).

[0051] Exemplary functional variants of E4Orf6 with removed splice sites: atgactacgtccggcgttccatttggcatgacactacgaccaacacgatctcggttgtctcggcgcactccgtacagtagggatcgcctacctccttttgagacagagacccgcgctaccatactggaggatcatccgctgctgcccgaatgtaacactttgacaatgcacaaTgtTTCCtacgtgcgaggtcttccctgcagtgtgggatttacgctgattcaggaatgggttgttccctgggatatggttctgacgcgggaggagcttgtaatcctgaggaagtgtatgcacgtgtgcctgtgttgtgccaacattgatatcatgacgagcatgatgatccatggttacgagtcctgggctctccactgtcattgttccagtcccggttccctgcagtgcatagccggcgggcaggttttggccagctggtttaggatggtggtggatggcgccatgtttaatcagaggtttatatggtaccgggaggtggtgaattacaacatgccaaaagaggtaatgtttatgtccagcgtgtttatgaggggtcgccacttaatctacctgcgcttgtggtatgatggccacgtgggttctgtggtccccgccatgagctttggatacagcgccttgcactgtgggattttgaacaatattgtggtgctgtgctgcagttactgtgctgatttaagtgagatcagggtgcgctgctgtgcccggaggacaaggcgtctcatgctgcgggcggtgcgaatcatcgctgaggagaccactgccatgttgtattcctgcaggacggagcggcggcggcagcagtttattcgcgcgctgctgcagcaccaccgccctatcctgatgcacgattatgactctacccccatgTAGtaa(SEQ ID NO: 23)

[0052] In some embodiments, viral vector production components comprise one or more polynucleotides that collectively encode the gene products required to generate lentiviral vectors in recombinant host cells.Exemplary lentiviral gene products include VSV-G, Gag-Pol and Rev.In some embodiments, viral vectors comprise one or more polynucleotides that collectively encode the gene products: VSV-G (or a functional variant thereof), Gag-Pol (or a functional variant thereof) and Rev (or a functional variant thereof).

[0053] In some embodiments, the viral vector components (i.e., the gene products of the viral vector components) are encoded on a single polynucleic acid. In other embodiments, multiple polynucleic acids collectively comprise the viral vector components (i.e., at least two of the gene products of the viral vector components are encoded on different polynucleic acids). For example, the viral vector components may comprise at least 2, at least 3, at least 4, or at least 5 polynucleic acids. In some embodiments, the viral vector components comprise 2, 3, 4, or 5 polynucleic acids.

[0054] In addition to the viral vector components, the viral vector production systems described herein include a first expression cassette that includes a nucleic acid sequence of a promoter operably linked to a nucleic acid sequence encoding a regulatory RNA.

[0055] As described in more detail below (parts IA and IB), exemplary regulatory RNAs include shRNAs and Cas13 guide RNAs.

[0056] The first expression cassette may comprise tandem repeats of a nucleic acid sequence encoding a regulatory RNA. For example, in some embodiments, the tandem repeats may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 copies of a nucleic acid sequence encoding a regulatory RNA. In some embodiments, the tandem repeats comprise 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 copies of a nucleic acid sequence encoding a regulatory RNA. In some embodiments, the tandem repeat comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a nucleic acid sequence encoding a regulatory RNA.

[0057] The first expression cassette may comprise two or more tandem repeats of a nucleic acid sequence encoding a regulatory RNA. For example, in some embodiments, the first expression cassette comprises at least 2, at least 3, at least 4, or at least 5 tandem repeats of a nucleic acid sequence encoding a regulatory RNA. In some embodiments, the first expression cassette comprises 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 tandem repeats of a nucleic acid sequence encoding a regulatory RNA. In some embodiments, the first expression cassette comprises 2, 3, 4, or 5 tandem repeats of a nucleic acid sequence encoding a regulatory RNA.

[0058] The first expression cassette may contain a cluster of nucleic acid sequences encoding regulatory RNAs (e.g., an shRNA cluster or an artificial miRNA cluster). As used herein, the term cluster refers to a set of two or more miRNAs that are physically adjacent (i.e., within about 10 kilobases), transcribed in the same direction, and not separated by a transcription unit, or a polynucleic acid that encodes one miRNA in opposite orientation, as described in Lai, X., and J. Vera, "MicroRNA clusters." Encyclopedia of Systems Biology. New York: Springer (2013), incorporated by reference in its entirety. Exemplary miRNA clusters are miR-30e, miR-30c-1, miR-214, miR-199a-2, miR-215, miR-194-1, miR-217, miR-216, miR-15b, miR-16-2, miR-143, miR-145, miR-25, miR-93, miR-106b, miR-23b, miR-27b, miR-24-1, miR-181a, miR-181b-2, miR-34b; miR-34c, miR-125b-1, let-7a-2, miR-100, miR-16-1 , miR-15a, miR-17, miR-18, miR-19a, miR-20, miR-19b-1, miR-92-1, miR-299, miR-323, miR-329, miR-134, miR-154, miR-133a-1, miR-1-2, miR-99b, let-7e, miR-125a, miR-133a-2, miR-1-1, miR-99a, let-7c, miR-125b-2, miR-98, let-7f-2, miR-105-1, and miR-105-2. In some embodiments, the cluster encodes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 miRNAs. An shRNA cluster or artificial miRNA cluster, as described herein, refers to a cluster in which the hairpin of an miRNA is replaced with a hairpin of an shRNA or artificial miRNA as described herein (e.g., an shRNA hairpin targeting a payload gene).Methods for producing amiRNA clusters are well known in the art and are described in Bhaskaran, Vivek, et al. Nature protocols 14.12 (2019): 3538-3553, which is incorporated by reference in its entirety. In some embodiments, the shRNA or amiRNA cluster comprises a non-naturally occurring shRNA or amiRNA hairpin. In some embodiments, the 5' and 3' most adjacent regions of the shRNA or amiRNA cluster are replaced with the adjacent regions of a different miRNA cluster to produce a chimeric shRNA or amiRNA cluster.

[0059] The first expression cassette may comprise nucleic acid sequences of different regulatory RNAs (i.e., regulatory RNAs having different nucleic acid sequences). For example, in some embodiments, the first expression cassette comprises nucleic acid sequences of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different regulatory RNAs. In some embodiments, the first expression cassette comprises nucleic acid sequences of 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 different regulatory RNAs. In some embodiments, the first expression cassette comprises nucleic acid sequences of 2, 3, 4, 5, 6, 7, 8, 9, or 10 different regulatory RNAs.

[0060] As described herein, a promoter is "operably linked" to a nucleic acid coding sequence when the position of the promoter relative to the nucleic acid coding sequence is such that binding of a transcriptional activator to the promoter can induce expression of the coding sequence.

[0061] The promoter may be a constitutive promoter (i.e., an unregulated promoter that allows continuous transcription). Examples of constitutive promoters are known in the art and include, but are not limited to, cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EF1α) promoter, simian vacuolating virus 40 (SV40) promoter, ubiquitin-C (UBC) promoter, U6 promoter, and phosphoglycerate kinase (PGK) promoter. For example, see Ferreira et al., Tuning gene expression with synthetic upstream open reading frames. Proc. Natl. Acad. Sci. USA 2013 July; 110(28): 11284-89; US Patent Application Publication No. 2014 / 377861; Qin, Jane Yuxia, et al., “PloS one 5.5 (2010): e10611, which are incorporated herein by reference in their entirety.

[0062] Alternatively, the promoter may be an inducible promoter (i.e., it activates transcription only under certain circumstances). The inducible promoter may be, for example, a chemically inducible promoter, a temperature inducible promoter, or a light inducible promoter. Examples of inducible promoters are known in the art and include, but are not limited to, tetracycline / doxycycline inducible promoters, coumarate inducible promoters, ABA inducible promoters, CRY2-CIB1 inducible promoters, DAPG inducible promoters, and mifepristone inducible promoters. For example, see Stanton et al., ACS Synth.Biol. 2014 Dec 19;3(12):880-91; Liang et al., Sci.Signal. 2011 Mar 15;4(164):rs2; Patent Nos.: U.S. Pat. No. 7,745,592; U.S. Pat. No. 7,935,788, which are incorporated herein by reference in their entirety.

[0063] In some embodiments, the promoter of the first expression cassette is a constitutive promoter, such as a CMV promoter, an EF1α promoter, an SV40 promoter, a UBC promoter, a U6 promoter, or a PGK promoter.

[0064] In some embodiments, the promoter of the first expression cassette is an inducible promoter, such as a chemically inducible promoter, a temperature inducible promoter, or a light inducible promoter. In some embodiments, the inducible promoter is a tetracycline / doxycycline inducible promoter, a coumarate inducible promoter, an ABA inducible promoter, a CRY2-CIB1 inducible promoter, a DAPG inducible promoter, or a mifepristone inducible promoter.

[0065] In some embodiments, the first expression cassette further comprises a nucleic acid sequence encoding the gene product of the viral vector production component described above. For example, in an embodiment in which the viral vector production component is an AAV viral vector component, the first expression cassette may further comprise a nucleic acid sequence encoding Rep52, Rep40, Rep78, Rep68, E2A, E4Orf6, VARNA, VP1, VP2, VP3, AAP, or a combination thereof. Similarly, in an embodiment in which the viral vector production component is a lentiviral vector component, the first expression cassette may further comprise a nucleic acid sequence encoding VSV-G, Gag-Pol, Rev, or a combination thereof.

[0066] In some embodiments, the promoter of the first expression cassette is operably linked to both a nucleic acid sequence encoding a regulatory RNA and a nucleic acid sequence encoding a gene product of a viral vector production component. For example, in some embodiments, the nucleic acid sequence encoding the gene product has a 5'UTR, an intron, and / or a 3'UTR that includes a nucleic acid sequence encoding a regulatory RNA.

[0067] In some embodiments, the first expression cassette further comprises a nucleic acid sequence encoding a selectable marker. As used herein, the term "selectable marker" refers to a protein that, when introduced into or expressed in a cell, confers a trait suitable for selection.

[0068] The selectable marker may be a fluorescent protein. Examples of fluorescent proteins are known in the art (e.g., TagBFP, EBFP2, EGFP, EYFP, mKO2, or Sirius). For example, see U.S. Patent No. 5,874,304; European Patent No. 0969284; U.S. Patent Application Publication No. 2010 / 167394, which are incorporated herein by reference in their entirety.

[0069] Alternatively, or in addition, the selectable marker may be an antibiotic resistance protein. Examples of antibiotic resistance proteins are known in the art (e.g., facilitating puromycin, hygromycin, neomycin, zeocin, blasticidin, or phleomycin selection). See, for example, Publication No. WO 1997 / 15668; Publication No. WO 1997 / 43900, which are incorporated herein by reference in their entireties.

[0070] In some embodiments, the promoter of the first expression cassette is operably linked to both a nucleic acid sequence encoding a regulatory RNA and a nucleic acid sequence encoding a selectable marker. For example, in some embodiments, the nucleic acid sequence encoding the selectable marker has a 5'UTR, an intron and / or a 3'UTR that includes a nucleic acid sequence encoding a regulatory RNA.

[0071] In addition to viral vector components and the first expression cassette, the viral vector production system described herein comprises a transfer polynucleic acid.The transfer polynucleic acid described herein comprises a central nucleic acid sequence flanked at 5' and 3' ends by the nucleic acid sequence of viral terminal repeat.As used herein, the term "viral terminal repeat" refers to the nucleic acid sequence required for polynucleic acid integration of viral vector payload into host cell genome.Exemplary viral terminal repeats are known to those skilled in the art.

[0072] For example, in embodiments in which the viral vector production component is an AAV viral vector component, the transferred polynucleic acid may comprise a central nucleic acid sequence flanked at the 5' and 3' ends by nucleic acid sequences of AAV inverted tandem repeats ("ITRs"). Exemplary AAV ITRs are known to those of skill in the art.

[0073] In embodiments in which the viral vector production component is a lentiviral vector component, the transferred polynucleic acid may comprise a central nucleic acid sequence flanked at the 5' and 3' ends by nucleic acid sequences of lentiviral long tandem repeats (LTRs). Exemplary lentiviral LTRs are known to those of skill in the art.

[0074] The central nucleic acid of the imported polynucleic acid may comprise a target nucleic acid sequence that is complementary to the regulatory RNA encoded by the first expression cassette. As described herein, a target nucleic acid sequence "complements" a regulatory RNA if it can be bound by (i.e., hybridize with) the regulatory RNA under physiological conditions of a host cell. A target nucleic acid sequence is said to have 100% complementarity to a regulatory RNA if it comprises a nucleic acid sequence that is the reverse complement of the regulatory RNA. In some embodiments, the target nucleic acid sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementarity to the regulatory RNA. In some embodiments, the target nucleic acid sequence has 85-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100% complementarity to the regulatory RNA.

[0075] Alternatively or in addition, the central nucleic acid of the transfer polynucleic acid may comprise a nucleic acid sequence of a multiple cloning site. Exemplary multiple cloning sites are known to those skilled in the art. The multiple cloning site may be used to clone a payload molecule (or gene of interest) - or an expression cassette encoding a payload molecule - into the transfer polynucleic acid before generating a viral vector in a host cell. In some embodiments, the nucleic acid sequence of the multiple cloning site is flanked (at the 5' end, the 3' end, or at both the 5' end and the 3' end) by a target nucleic acid sequence that is complementary to the regulatory RNA encoded by the first expression cassette, all contained within the central nucleic acid. For example, the central nucleic acid may comprise a 5'UTR sequence and / or a 3'UTR sequence that comprises a target nucleic acid sequence (complementary to the regulatory RNA encoded by the first expression cassette) that may be operably linked to a gene of interest cloned into the multiple cloning site. In some embodiments, the central nucleic acid further comprises a nucleic acid sequence of a promoter (constitutive or inducible as described herein). For example, in some embodiments, the imported polynucleic acid comprises, from 5' to 3': (i) a nucleic acid sequence of a viral terminal repeat; (ii) a nucleic acid sequence of a promoter; (iii) a nucleic acid sequence of a multiple cloning site flanked (at the 3' end, the 5' end, or both the 5' end and the 3' end) by targeting nucleic acid sequences that are complementary to a regulatory RNA encoded by the first expression cassette; and (iv) a nucleic acid sequence of a viral terminal repeat.

[0076] Alternatively, or in addition, the central nucleic acid of the imported polynucleic acid may comprise an expression cassette comprising a promoter (constitutive or inducible as described herein) and a target nucleic acid sequence complementary to the regulatory RNA encoded by the first expression cassette, both of which are operably linked to a nucleic acid sequence encoding a payload molecule. In some embodiments, the nucleic acid sequence encoding a payload molecule comprises a 5'UTR comprising a target nucleic acid sequence; a coding sequence comprising a target nucleic acid sequence; a 3'UTR comprising a target nucleic acid sequence; or a combination thereof.

[0077] The central nucleic acid may comprise tandem repeats of the target nucleic acid sequence (i.e., a nucleic acid sequence complementary to the regulatory RNA encoded by the first expression cassette). In some embodiments, the tandem repeats may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 copies of the target nucleic acid sequence. In some embodiments, the tandem repeats comprise 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 copies of the target nucleic acid sequence. In some embodiments, the tandem repeat comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 copies of a target nucleic acid sequence.

[0078] The central nucleic acid may comprise two or more tandem repeats of a target nucleic acid sequence (i.e., a nucleic acid sequence complementary to a regulatory RNA encoded by the first expression cassette). In some embodiments, the central nucleic acid comprises at least 2, at least 3, at least 4, or at least 5 tandem repeats of a nucleic acid sequence encoding a regulatory RNA. In some embodiments, the central nucleic acid comprises 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 tandem repeats of a nucleic acid sequence encoding a regulatory RNA. In some embodiments, the central nucleic acid comprises 2, 3, 4, or 5 tandem repeats of a nucleic acid sequence encoding a regulatory RNA.

[0079] In embodiments in which the first expression cassette comprises distinct regulatory RNAs (i.e., regulatory RNAs having different nucleic acid sequences), the central nucleic acid may comprise distinct target nucleic acid sequences. For example, in some embodiments, the central nucleic acid comprises nucleic acid sequences of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 distinct target nucleic acid sequences. In some embodiments, the central nucleic acid comprises nucleic acid sequences of 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 distinct target nucleic acid sequences. In some embodiments, the central nucleic acid comprises nucleic acid sequences of 2, 3, 4, 5, 6, 7, 8, 9 or 10 different target nucleic acid sequences.

[0080] In some embodiments, the viral vector production systems described herein comprise engineered cells. The engineered cells may comprise any part (and any combination of parts) of the viral vector production systems described herein.

[0081] For example, the engineered cell may comprise at least a portion of the viral vector production components. For example, as described above, the viral vector production components may comprise a plurality of polynucleic acids. In such an embodiment, the engineered cell comprises one or more of the plurality of polynucleic acids, each of which may be located extrachromosomally or stably integrated into the genome of the engineered cell. In some embodiments, the engineered cell comprises the entire viral vector production components.

[0082] Alternatively, or in addition, the engineered cell may contain a first expression cassette for a viral production system.

[0083] Alternatively, or in addition, the engineered cells may contain a transfected polynucleic acid of a viral production system.

[0084] In some embodiments, a viral vector production system comprises: (a) an engineered cell comprising viral vector production components that comprise one or more heterologous polynucleic acids that collectively encode gene products of a viral vector; (b) a heterologous nucleic acid sequence encoding a first expression cassette, the first expression cassette comprising a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a regulatory RNA; and (c) an imported polynucleic acid comprising a central nucleic acid sequence flanked at the 5' and 3' ends by nucleic acid sequences of viral terminal repeats.

[0085] A. Production system with shRNA regulatory RNA. In some embodiments, the regulatory RNA of the viral vector production system is an shRNA. A small hairpin RNA ("shRNA") is a sequence that mimics a microRNA and downregulates RNA transcripts that have sufficient complementarity to the microRNA sequence. shRNAs are transcribed by Pol II or Pol III promoters, processed, and incorporated into the RNA-induced silencing complex (RISC). In some embodiments, the regulatory RNA of the viral vector production system is an amiRNA (artificial microRNA). Artificial miRNAs are naturally occurring pri-miRNA sequences that are modified to include sequences that direct the downregulation of a target gene (e.g., a payload gene). shRNAs and amiRNAs can be designed to be complementary to the payload molecule (GOI) coding sequence and / or UTR (5'UTR or 3'UTR). A specific target sequence can be incorporated, for example, into the UTR sequence of the payload molecule.

[0086] A production system having an shRNA as a regulatory RNA may have any of the above aspects (Part I). A production system having an amiRNA as a regulatory RNA may have any of the above aspects (Part I).

[0087] In some embodiments, the nucleic acid sequence encoding the shRNA comprises any one of the nucleic acid sequences set forth in SEQ ID NOs: 2 to 11. In some embodiments, the shRNA or amiRNA targets any one of the sequences set forth in SEQ ID NOs: 13 to 22.

[0088] In some embodiments, the first expression cassette comprises a nucleic acid sequence encoding a selectable marker (described herein), wherein the nucleic acid sequence encoding the selectable marker comprises from 5' to 3' an intron having: (i) an intron donor site; (ii) a nucleic acid sequence encoding an shRNA; and (iii) an intron acceptor site.

[0089] In some embodiments, the shRNA is operably linked to a PolIII promoter (e.g., a U6 promoter). In some embodiments, the amiRNA is operably linked to a PolIII promoter (e.g., a U6 promoter).

[0090] In some embodiments, the intron comprises tandem repeats of a nucleic acid sequence encoding an shRNA. In some embodiments, the tandem repeat comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a nucleic acid sequence encoding an shRNA. In some embodiments, the tandem repeat comprises at least 2, at least 3, at least 4, or at least 5 copies of a nucleic acid sequence encoding an shRNA. In some embodiments, the tandem repeat comprises 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 copies of a nucleic acid sequence encoding an shRNA.

[0091] In some embodiments, the intron comprises an shRNA cluster of a nucleic acid sequence encoding an shRNA. In some embodiments, the shRNA cluster comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a nucleic acid sequence encoding an shRNA. In some embodiments, the shRNA cluster comprises at least 2, at least 3, at least 4, or at least 5 copies of a nucleic acid sequence encoding an shRNA. In some embodiments, the shRNA cluster comprises 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 copies of a nucleic acid sequence encoding an shRNA.

[0092] In some embodiments, the intron comprises an amiRNA cluster of nucleic acid sequences encoding the amiRNA. In some embodiments, the amiRNA cluster comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the nucleic acid sequence encoding the amiRNA. In some embodiments, the amiRNA cluster comprises at least 2, at least 3, at least 4, or at least 5 copies of the nucleic acid sequence encoding the amiRNA. In some embodiments, the amiRNA cluster comprises 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 copies of the nucleic acid sequence encoding the amiRNA.

[0093] In some embodiments, the nucleic acid sequence encoding the selectable marker comprises a 5'UTR, where the intron of the selectable marker is located in the 5'UTR; a 3'UTR, where the intron of the selectable marker is located in the 3'UTR; or a combination thereof. In some embodiments, the intron of the selectable marker is located in the coding region of the nucleic acid sequence encoding the selectable marker.

[0094] In some embodiments, the viral vector production system is as shown in FIG.

[0095] In some embodiments, the intron comprises a nucleic acid sequence of AGgtaagtNNNNTACTTTAGGACCCTTTTTTTTCCacagGT (SEQ ID NO: 12), where "NNNN" comprises a targeting sequence of the shRNA. In some embodiments, "NNNN" comprises any one of SEQ ID NOs: 2-11.

[0096] B. Production system with Cas13 guide RNA regulatory RNA. In some embodiments, the regulatory RNA of the viral vector production system is a Cas13 guide RNA. Cas13 is a programmable RNA guide RNA targeting Cas protein with nuclease activity that allows target mRNA knockdown without changing the coding DNA sequence of the gene. Cas13 is guided to the target RNA by a guide RNA that is complementary to the target sequence. Target recognition results in RNA-RNA hybridization and cleavage of the target RNA. The guide RNA can be designed to be complementary to the payload molecule (GOI) coding sequence and / or UTR (5'UTR or 3'UTR). A specific target sequence can be incorporated, for example, into the UTR sequence of the payload molecule. Cas13 can be used to knock down the payload molecule ("GOI") protein level by RNA cleavage without modifying the coding DNA sequence. Cas13 does not exhibit a protospacer adjacent sequence requirement that allows targeting of any sequence within the transcribed region.

[0097] A production system having a Cas13 guide RNA as a regulatory RNA may have any of the above aspects (part I).

[0098] In some embodiments, the first expression cassette comprises a constitutive promoter operably linked to a nucleic acid sequence encoding two or more different Cas13 guide RNAs. In some embodiments, the two or more different gRNAs are included in a guide RNA array selected from the group consisting of a native guide RNA array, a ribozyme self-cleaving guide RNA array, a Cys4 guide RNA array, or a tRNA guide RNA array as described in McCarty, Nicholas S., et al., “Nature communications 11.1 (2020): 1-13, the entirety of which is incorporated by reference. In some embodiments, the first expression cassette comprises a nucleic acid sequence encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 different Cas13 guide RNAs. In some embodiments, the first expression cassette comprises at least 2, at least 3, at least 4, or at least 5 different Cas13 guide RNAs. In some embodiments, the first expression cassette comprises a nucleic acid sequence encoding 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 different Cas13 guide RNAs.

[0099] In some embodiments, the viral vector production system further comprises a heterologous polynucleic acid encoding Cas13. Exemplary Cas13 proteins are known to those of skill in the art and include, but are not limited to, Cas13a, Cas13b, Cas13c, and Cas13d. In some embodiments, the viral vector production system comprises a heterologous polynucleic acid encoding Cas13a. In some embodiments, the viral vector production system comprises a heterologous polynucleic acid encoding Cas13b. In some embodiments, the viral vector production system comprises a heterologous polynucleic acid encoding Cas13c. In some embodiments, the viral vector production system comprises a heterologous polynucleic acid encoding Cas13d.

[0100] In some embodiments, Cas13 comprises the amino acid sequence of SEQ ID NO: 1 or a functional variant having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the amino acid sequence of SEQ ID NO: 1. As used herein, the term "functional variant" in the context of a Cas13 protein refers to a variant that has at least 50% of the endonuclease activity compared to a wild-type Cas13 protein.

[0101] Methods for determining the degree of identity between two sequences (e.g., two amino acid sequences or two polynucleic acids) are known to those skilled in the art. One exemplary method is the use of the Basic Local Alignment Search Tool (BLAST®) software with default parameters (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0102] In some embodiments, the heterologous polynucleic acid encoding Cas13 further comprises a first expression cassette. In some embodiments, the first expression cassette comprises a nucleic acid sequence of a constitutive promoter operably linked to both a nucleic acid sequence encoding a Cas13 gRNA and a nucleic acid sequence encoding Cas13.

[0103] In some embodiments, the engineered cells contain a heterologous polynucleic acid encoding Cas13.

[0104] In some embodiments, the viral vector production system is as shown in FIG.

[0105] II. Engineered Cells for Viral Vector Production In some aspects, the present disclosure relates to engineered cells for viral vector production. These engineered cells may include any combination of parts of the viral vector production system described above. Exemplary engineered cells for viral vector production are provided below.

[0106] A. Engineered cells containing shRNA regulatory RNA. In some embodiments, an engineered cell for viral vector production comprises one or more heterologous polynucleic acids that collectively comprise: (a) viral vector production components that collectively encode a gene product of the viral vector; (b) a first expression cassette comprising a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding an shRNA; and (c) an import polynucleic acid comprising a central nucleic acid sequence flanked at the 5' and 3' ends by nucleic acid sequences of viral terminal repeats, the central nucleic acid sequence comprising a nucleic acid sequence encoding a payload molecule operably linked to both a promoter and a target nucleic acid sequence that is complementary to the shRNA encoded by the first expression cassette.

[0107] In some embodiments, the nucleic acid sequence of the payload molecule comprises a 5'UTR comprising a target nucleic acid sequence that is complementary to the shRNA encoded by the first expression cassette; a 3'UTR comprising a target nucleic acid sequence that is complementary to the shRNA encoded by the first expression cassette; or a combination thereof.

[0108] In some embodiments, the nucleic acid sequence of the payload molecule comprises a tandem repeat of a target nucleic acid sequence complementary to the shRNA encoded by the first expression cassette. In some embodiments, the tandem repeat may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 copies of the target nucleic acid sequence. In some embodiments, the tandem repeat comprises 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 copies of the target nucleic acid sequence. In some embodiments, the tandem repeat comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 copies of a target nucleic acid sequence.

[0109] In some embodiments, the first expression cassette comprises a tandem repeat of a nucleic acid sequence encoding an shRNA. In some embodiments, the tandem repeat comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a nucleic acid sequence encoding an shRNA. In some embodiments, the tandem repeat comprises at least 2, at least 3, at least 4, or at least 5 copies of a nucleic acid sequence encoding an shRNA. In some embodiments, the tandem repeat comprises 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 copies of a nucleic acid sequence encoding an shRNA.

[0110] In some embodiments, the nucleic acid sequence encoding the shRNA comprises any of the nucleic acid sequences of SEQ ID NOs: 2-11.

[0111] In some embodiments, the first expression cassette comprises nucleic acid sequences of two or more different shRNAs. For example, in some embodiments, the first expression cassette comprises nucleic acid sequences encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 different shRNAs. In some embodiments, the first expression cassette comprises nucleic acid sequences encoding at least 2, at least 3, at least 4, or at least 5 different shRNAs. In some embodiments, the first expression cassette comprises nucleic acid sequences encoding 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 different shRNAs.

[0112] In some embodiments, the first expression cassette comprises a nucleic acid sequence encoding a selectable marker, the nucleic acid sequence encoding the selectable marker comprising, from 5' to 3': (i) an intron donor site; (ii) a nucleic acid sequence encoding an shRNA; and (iii) an intron acceptor site.

[0113] In some embodiments, the intron comprises tandem repeats of a nucleic acid sequence encoding an shRNA.

[0114] In some embodiments, the nucleic acid sequence encoding the selectable marker comprises a 5'UTR, where the intron of the selectable marker is located in the 5'UTR; a 3'UTR, where the intron of the selectable marker is located in the 5'UTR; or a combination thereof. In some embodiments, the intron of the selectable marker is located in the coding region of the nucleic acid sequence encoding the selectable marker.

[0115] In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO:12.

[0116] In some embodiments, the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of an AAV vector. In some embodiments, the viral vector components comprise the nucleic acid sequences of Rep52 or Rep40; Rep78 or Rep68; E2A; E4Orf6; VARNA; VP1; VP2; VP3; and AAP. In some embodiments, the viral terminal repeat of the imported polynucleic acid is an AAV inverted tandem repeat.

[0117] In some embodiments, the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of lentiviral vector.In some embodiments, the viral vector components comprise the nucleic acid sequences of VSV-G, Gag-Pol and Rev.In some embodiments, the viral terminal repeat of the imported polynucleic acid is a lentiviral long terminal repeat.

[0118] In some embodiments, the engineered cells comprise a viral vector production system as shown in FIG.

[0119] In some embodiments, at least one of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell. In some embodiments, each of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell.

[0120] In some embodiments, the engineered cells are derived from HEK293 cells or HeLa cells, BHK cells or Sf9 cells.

[0121] B. Engineered cells containing Cas13 guide RNA regulatory RNA. In some embodiments, engineered cells for viral vector production comprise one or more heterologous polynucleic acids that collectively comprise: (a) viral vector production components that collectively encode gene products of the viral vector; (b) a first expression cassette comprising a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a Cas13 guide RNA; (c) a nucleic acid sequence encoding Cas13; and (d) an import polynucleic acid comprising a central nucleic acid sequence flanked at 5' and 3' ends by nucleic acid sequences of viral terminal repeats, the central nucleic acid sequence comprising a nucleic acid sequence encoding a payload molecule operably linked to both a promoter and a target nucleic acid sequence complementary to the Cas13 guide RNA encoded by the first expression cassette.

[0122] In some embodiments, the first expression cassette comprises a constitutive promoter operably linked to a nucleic acid sequence encoding two or more Cas13 guide RNAs. For example, in some embodiments, the first expression cassette comprises a nucleic acid sequence encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 different Cas13 guide RNAs. In some embodiments, the first expression cassette comprises a nucleic acid sequence encoding at least 2, at least 3, at least 4, or at least 5 different Cas13 guide RNAs. In some embodiments, the first expression cassette comprises a nucleic acid sequence encoding 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 different Cas13 guide RNAs.

[0123] In some embodiments, the viral vector production system comprises a heterologous polynucleic acid encoding Cas13a. In some embodiments, the viral vector production system comprises a heterologous polynucleic acid encoding Cas13b. In some embodiments, the viral vector production system comprises a heterologous polynucleic acid encoding Cas13c. In some embodiments, the viral vector production system comprises a heterologous polynucleic acid encoding Cas13d.

[0124] In some embodiments, the Cas13 comprises the amino acid sequence of SEQ ID NO:1, or the amino acid sequence of a functional variant having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the amino acid sequence of SEQ ID NO:1.

[0125] In some embodiments, the first expression cassette further comprises a nucleic acid sequence encoding Cas13. In some embodiments, the first expression cassette comprises a nucleic acid sequence of a constitutive promoter operably linked to both the nucleic acid sequence encoding the Cas13 gRNA and the nucleic acid sequence encoding Cas13.

[0126] In some embodiments, the nucleic acid sequence of the payload molecule comprises a 5'UTR comprising a target nucleic acid sequence that is complementary to the Cas13 guide RNA encoded by the first expression cassette; a 3'UTR comprising a target nucleic acid sequence that is complementary to the Cas13 guide RNA encoded by the first expression cassette; or a combination thereof.

[0127] In some embodiments, the nucleic acid sequence of the payload molecule comprises a tandem repeat of a target nucleic acid sequence complementary to the Cas13 guide RNA encoded by the first expression cassette. In some embodiments, the tandem repeat may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 copies of the target nucleic acid sequence. In some embodiments, the tandem repeat comprises 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 copies of the target nucleic acid sequence. In some embodiments, the tandem repeat comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 copies of a target nucleic acid sequence.

[0128] In some embodiments, the first expression cassette comprises tandem repeats of a nucleic acid sequence encoding a Cas13 guide RNA. In some embodiments, the tandem repeats may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 copies of a nucleic acid sequence encoding a Cas13 guide RNA. In some embodiments, the tandem repeats comprise 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 copies of a nucleic acid sequence encoding a Cas13 guide RNA. In some embodiments, the tandem repeat comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 copies of a nucleic acid sequence encoding a Cas13 guide RNA.

[0129] In some embodiments, the first expression cassette comprises nucleic acid sequences of two or more different Cas13 guide RNAs. For example, in some embodiments, the first expression cassette comprises nucleic acid sequences encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 different Cas13 guide RNAs. In some embodiments, the first expression cassette comprises nucleic acid sequences encoding at least 2, at least 3, at least 4, or at least 5 different Cas13 guide RNAs. In some embodiments, the first expression cassette comprises a nucleic acid sequence encoding 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, or 6-10 different Cas13 guide RNAs.

[0130] In some embodiments, the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of an AAV vector. In some embodiments, the viral vector components comprise the nucleic acid sequences of Rep52 or Rep40; Rep78 or Rep68; E2A; E4Orf6; VARNA; VP1; VP2; VP3; and AAP. In some embodiments, the viral terminal repeat of the imported polynucleic acid is an AAV inverted tandem repeat.

[0131] In some embodiments, the viral vector production components comprise one or more polynucleic acids that collectively encode the gene products of lentiviral vector.In some embodiments, the viral vector components comprise the nucleic acid sequences of VSV-G, Gag-Pol and Rev.In some embodiments, the viral terminal repeat of the imported polynucleic acid is a lentiviral long terminal repeat.

[0132] In some embodiments, the engineered cells comprise a viral vector production system as shown in FIG.

[0133] In some embodiments, at least one of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell. In some embodiments, each of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell.

[0134] In some embodiments, the engineered cells are derived from HEK293 cells or HeLa cells.

[0135] III. Methods for reducing payload expression during viral vector production In some aspects, the disclosure relates to methods of reducing expression of a payload during viral vector production utilizing an engineered cell as described in Part II, wherein the payload comprises a target nucleic acid sequence (complementary to a regulatory RNA encoded by a first expression cassette).

[0136] In some embodiments, where the engineered cells contain an shRNA as the regulatory RNA, a method of decreasing expression of the payload includes expressing the shRNA during viral vector production.

[0137] In some embodiments, where the engineered cells comprise a nucleic acid sequence encoding Cas13 and a nucleic acid sequence encoding a Cas13 guide RNA, methods of reducing expression of a payload include expressing Cas13 and the Cas13 guide RNA during viral vector production.

[0138] example Example 1. shRNA-mediated knockdown of AAV payload molecules. AAV pHelper, AAV pRepCap, and transfer plasmids were co-transfected into HEK293FT cells with or without an shRNA expression plasmid against EGFP. The three different shRNA plasmids were pooled together prior to testing (Figure 1). 72 hours after transfection, AAV was harvested by three freeze-thaw cycles in a dry ice isopropanol bath. The virus stock was serially diluted 1x, 10x, and 100x, and 10uL of the resulting virus stock was transduced by adding to 5e4 HEK293FT cells seeded in a 96-well plate. 48 hours after transduction, transduced cells were harvested and the percentage of EGFP positive cells was determined by flow cytometry, which was used to calculate transducing units / mL (TU / mL). (Figure 2A-B). Addition of EGFP shRNA resulted in a significant decrease in EGFP fluorescence and only a ~1.1-fold decrease in AAV titer. EGFP had minimal toxicity and the minimal drop in AAV titer indicated that the shRNA had minimal effects on AAV production and packaging, so AAV titer was not increased. Similar results were obtained when using FF5 shRNA with the FF5 target site on the transfer plasmid, along with a different immunoglobulin (IG)-EYFP transfer sequence (Figure 3). [Table 1-1] [Table 1-2] [Table 2]

[0139] Example 2. Cas13-mediated knockdown of AAV payload molecules. Cas13d can be used to knock down AAV payload GOI protein levels by RNA cleavage without modifying the GOI coding DNA sequence (Figure 4). Cas13d does not exhibit a protospacer flanking sequence requirement, allowing targeting of any sequence within the transcribed region.

[0140] Exemplary amino acid sequence of Cas13 (SEQ ID NO:1): 。

[0141] Other Aspects All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0142] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to adapt it to various applications and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the claims.

[0143] Equivalent Although several inventive embodiments have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is encompassed within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0144] All definitions and those used herein should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0145] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may cover the entire document.

[0146] The indefinite articles "a" and "an," as used herein and in the claims, unless clearly indicated otherwise, should be understood to mean "at least one."

[0147] The term "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may refer in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0148] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be judged to be inclusive, i.e., including at least one of, but also including more than one, and optionally including additional unlisted items, of a number or list of elements. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein, when used in the claims, when preceded by terms of exclusivity, such as "either," "one of," "only one of," "exactly one of," "consisting essentially of," etc., shall be judged only as indicating exclusive alternatives (i.e., "one or the other but not both") and shall have its ordinary meaning as used in the field of patent law.

[0149] As used herein and in the claims, the phrase "at least one" in reference to a list of one or more elements means at least one element selected from any one or more of the elements in the list of elements, but it should be understood that it does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally including more than one, A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one, B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (optionally including other elements), etc.

[0150] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0151] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., meaning inclusive but not exclusive. Only the transitional phrases "consisting of" and "consisting essentially of" are intended to be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It is to be understood that embodiments described herein using open-ended transitional phrases (e.g., "comprising") are also contemplated in alternative embodiments as "consisting of" and "consisting essentially of" the features described by the open-ended transitional phrases. For example, if the disclosure describes "a composition comprising A and B," the disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."

Claims

1. A viral vector production system comprising: (a) an engineered cell comprising a viral vector production component comprising one or more heterologous polynucleic acids collectively encoding the gene products of a viral vector; (b) a heterologous nucleic acid sequence encoding a first expression cassette, the first expression cassette comprising a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a regulatory RNA; and (c) a transfer polynucleic acid comprising a central nucleic acid sequence flanked at the 5' and 3' ends by nucleic acid sequences of viral terminal repeats The viral vector production system.

2. The viral vector production system according to claim 1, wherein the central nucleic acid sequence of the transfer polynucleic acid comprises a nucleic acid sequence encoding a multiple cloning site, Optionally, the central nucleic acid sequence comprises a multiple cloning sequence flanked by a target nucleic acid sequence complementary to the regulatory RNA encoded by the first expression cassette, Further optionally, the multiple cloning sequence is flanked by tandem repeats of a target nucleic acid sequence complementary to the regulatory RNA encoded by the first expression cassette, Further optionally, the multiple cloning sequence is flanked at the 5' and 3' ends by a target nucleic acid sequence complementary to the regulatory RNA encoded by the first expression cassette, said viral vector production system.

3. The viral vector production system according to claim 1 or 2, wherein The central nucleic acid sequence further comprises a promoter, and / or The central nucleic acid sequence of the transfer polynucleic acid sequence comprises a second expression cassette, the second expression cassette comprising a nucleic acid sequence encoding a payload molecule operably linked to both a promoter and a target nucleic acid sequence complementary to the regulatory RNA encoded by the first expression cassette, optionally, the nucleic acid sequence of the payload molecule comprises a 5'UTR comprising a target nucleic acid sequence complementary to the regulatory RNA encoded by the first expression cassette; a 3'UTR comprising a target nucleic acid sequence complementary to the regulatory RNA encoded by the first expression cassette; or a combination thereof, further optionally, the nucleic acid sequence of the payload molecule comprises tandem repeats of a target nucleic acid sequence complementary to the regulatory RNA encoded by the first expression cassette, and / or The first expression cassette comprises tandem repeats of a nucleic acid sequence encoding a regulatory RNA, and / or The first expression cassette comprises nucleic acid sequences of two or more different regulatory RNAs, and / or, The first expression cassette further comprises a nucleic acid sequence encoding a gene product of a viral vector production component. Optionally, the nucleic acid sequence encoding the gene product in the first expression cassette has a 5' UTR comprising a nucleic acid sequence encoding a regulatory RNA; an intron comprising a nucleic acid sequence encoding a regulatory RNA; a 3' UTR comprising a nucleic acid sequence encoding a regulatory RNA; or a combination thereof, and / or, The first expression cassette further comprises a nucleic acid sequence encoding a selectable marker. Optionally, the selectable marker comprises a fluorescent protein or an antibiotic resistance protein. Further optionally, the nucleic acid sequence encoding the selectable marker in the first expression cassette has a 5' UTR comprising a nucleic acid sequence encoding a regulatory RNA; an intron comprising a nucleic acid sequence encoding a regulatory RNA; a 3' UTR comprising a nucleic acid sequence encoding a regulatory RNA; or a combination thereof. The viral vector production system.

4. The viral vector production system according to claim 1 or 2, wherein (a) the viral vector production system is an AAV viral vector production system, and the viral vector production component comprises one or more polynucleotides collectively encoding gene products of the AAV vector. Optionally, the viral vector component comprises the nucleic acid sequences of Rep52 or Rep40; Rep78 or Rep68; E2A; E4Orf6; VARNA; VP1; VP2; VP3; and AAP. Further optionally, the viral terminal repeats of the transduced polynucleotide are AAV inverted tandem repeats, or, (b) the viral vector production system is a lentiviral vector production system, and the viral vector production component comprises one or more polynucleotides collectively encoding gene products of the lentiviral vector. Optionally, the viral vector component comprises the nucleic acid sequences of VSV-G, Gag-Pol and Rev. Further optionally, the viral terminal repeats of the transduced polynucleotide are lentiviral long terminal repeats. The viral vector production system.

5. At least one of the one or more heterologous polynucleotides of the viral vector production component is stably integrated into the genome of the engineered cell, and / or, each of one or more heterologous polynucleic acids of the viral vector production components is stably integrated into the genome of the engineered cell, and / or the engineered cell further comprises a heterologous nucleic acid sequence encoding a first expression cassette, and / or the engineered cell is derived from HEK293 cells, HeLa cells, BHK cells or Sf9 cells, the viral vector production system according to claim 1 or 2.

6. the regulatory RNA is shRNA or amiRNA, optionally, the nucleic acid sequence encoding shRNA comprises any one of the nucleic acid sequences of SEQ ID NOs: 2-11, further optionally, the first expression cassette comprises a nucleic acid sequence encoding a selectable marker, and the nucleic acid sequence encoding the selectable marker is, from 5' to 3': (i) an intron donor site; (ii) a nucleic acid sequence encoding shRNA or amiRNA; and (iii) an intron containing an intron acceptor site, optionally, the intron comprises a tandem repeat of a nucleic acid sequence encoding shRNA or amiRNA, a shRNA cluster or an amiRNA cluster, further optionally, the nucleic acid sequence encoding the selectable marker is a 5'UTR, and the intron of the selectable marker is located in the 5'UTR; a 3'UTR, and the intron of the selectable marker is located in the 3'UTR; or a combination thereof, further optionally, the intron of the selectable marker is located in the coding region of the nucleic acid sequence encoding the selectable marker, further optionally, the intron comprises the nucleic acid sequence of SEQ ID NOs: 12, the viral vector production system according to claim 1 or 2.

7. the regulatory RNA is a Cas13 guide RNA, the viral vector production system according to claim 1 or 2, optionally, the first expression cassette comprises a constitutive promoter operably linked to a nucleic acid sequence encoding two or more Cas13 guide RNAs, and / or optionally, further comprises a heterologous polynucleic acid encoding Cas13, further optionally, (a) Cas13 is Cas13d, optionally, Cas13d comprises the amino acid sequence of SEQ ID NOs: 1, or an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NOs: 1, and / or (b) The heterologous polynucleic acid encoding Cas13 further comprises a first expression cassette, optionally, the first expression cassette comprises a nucleic acid sequence of a constitutive promoter operably linked to both a nucleic acid sequence encoding a Cas13 gRNA and a nucleic acid sequence encoding Cas13, and / or, (c) The engineered cell further comprises a heterologous polynucleic acid encoding Cas13, said viral vector production system. **Claim 8** (a) Viral vector production components that collectively encode the gene products of a viral vector; (b) A first expression cassette comprising a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding an shRNA or an amiRNA; and (c) A transfer polynucleic acid comprising a central nucleic acid sequence flanked at the 5' and 3' ends by nucleic acid sequences of viral terminal repeats, the central nucleic acid sequence comprising a nucleic acid sequence encoding a payload molecule operably linked to both a promoter and a target nucleic acid sequence complementary to the shRNA or amiRNA encoded by the first expression cassette, the transfer polynucleic acid An engineered cell for the production of a viral vector, comprising one or more heterologous polynucleic acids collectively comprising the above. **Claim 9** The nucleic acid sequence of the payload molecule comprises a 5' UTR comprising a target nucleic acid sequence complementary to the shRNA or amiRNA encoded by the first expression cassette; a 3' UTR comprising a target nucleic acid sequence complementary to the shRNA or amiRNA encoded by the first expression cassette; or a combination thereof, and / or, The nucleic acid sequence of the payload molecule comprises a tandem repeat of a target nucleic acid sequence complementary to the shRNA or amiRNA encoded by the first expression cassette, and / or, The first expression cassette comprises a tandem repeat of a nucleic acid sequence encoding an shRNA or an amiRNA, an shRNA cluster or an amiRNA cluster, and / or, The first expression cassette comprises nucleic acid sequences of two or more different shRNAs or two or more different amiRNAs, the engineered cell according to claim 8. **Claim 10** The first expression cassette comprises a nucleic acid sequence encoding a selectable marker, and the nucleic acid sequence encoding the selectable marker has, from 5' to 3': (i) an intron donor site; (ii) a nucleic acid sequence encoding an shRNA or an amiRNA; and (iii) an intron having an intron acceptor site, the engineered cell according to claim 8 or 9, Optionally, the intron comprises a tandem repeat of a nucleic acid sequence encoding an shRNA or an amiRNA, an shRNA cluster or an amiRNA cluster, Further optionally, the nucleic acid sequence encoding the selectable marker is a 5'UTR, wherein the intron of the selectable marker is located in the 5'UTR; a 3'UTR, wherein the intron of the selectable marker is located in the 3'UTR; or a combination thereof, Further optionally, the intron of the selectable marker is located in the coding region of the nucleic acid sequence encoding the selectable marker, Further optionally, the intron comprises the nucleic acid sequence of SEQ ID NO: 12, the engineered cell.

11. The nucleic acid sequence encoding the shRNA comprises any one of the nucleic acid sequences of SEQ ID NOs: 2 to 11, and / or, (a) The viral vector production component comprises one or more polynucleotides collectively encoding the gene products of the AAV vector, optionally, the viral vector component comprises the nucleic acid sequences of Rep52 or Rep40; Rep78 or Rep68; E2A; E4Orf6; VARNA; VP1; VP2; VP3; and AAP, further optionally, the viral terminal repeats of the transferred polynucleotide are AAV inverted tandem repeats, or, (b) The viral vector production component comprises one or more polynucleotides collectively encoding the gene products of the lentiviral vector, optionally, the viral vector component comprises the nucleic acid sequences of VSV-G, Gag-Pol and Rev, further optionally, the viral terminal repeats of the transferred polynucleotide are lentiviral long terminal repeats, the engineered cell according to claim 8 or claim 9.

12. At least one of the one or more heterologous polynucleotides is stably integrated into the genome of the engineered cell, and / or, Each of the one or more heterologous polynucleotides is stably integrated into the genome of the engineered cell, and / or, The engineered cell according to claim 8 or 9, wherein the engineered cell is derived from HEK293 cells, HeLa cells, BHK cells or Sf9 cells.

13. (a) A viral vector production component that collectively encodes gene products of a viral vector; (b) A first expression cassette comprising a nucleic acid sequence of a constitutive promoter operably linked to a nucleic acid sequence encoding a Cas13 guide RNA; (c) A nucleic acid sequence encoding Cas13; and (d) A transfer polynucleotide comprising a central nucleic acid sequence flanked at the 5' and 3' ends by nucleic acid sequences of viral terminal repeats, the central nucleic acid sequence being operably linked to both a target nucleic acid sequence complementary to a Cas13 guide RNA encoded by a promoter and the first expression cassette A transfer polynucleotide comprising a nucleic acid sequence encoding a payload molecule. An engineered cell for viral vector production, comprising one or more heterologous polynucleotides collectively comprising the above.

14. The engineered cell according to claim 13, wherein the first expression cassette comprises a constitutive promoter operably linked to a nucleic acid sequence encoding two or more Cas13 guide RNAs, and / or Cas13 is Cas13d, optionally, Cas13d comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1, and / or the first expression cassette further comprises a nucleic acid sequence encoding Cas13, optionally, the first expression cassette comprises a nucleic acid sequence encoding a Cas13 gRNA and a nucleic acid sequence of a constitutive promoter operably linked to both nucleic acid sequences encoding Cas13, and / or the nucleic acid sequence of the payload molecule comprises a 5'UTR comprising a target nucleic acid sequence complementary to a Cas13 guide RNA encoded by the first expression cassette; a 3'UTR comprising a target nucleic acid sequence complementary to a Cas13 guide RNA encoded by the first expression cassette; or a combination thereof, and / or the nucleic acid sequence of the payload molecule comprises a tandem repeat of a target nucleic acid sequence complementary to a Cas13 guide RNA encoded by the first expression cassette, and / or the first expression cassette comprises a tandem repeat of a nucleic acid sequence encoding a Cas13 guide RNA, and / or The first expression cassette comprises nucleic acid sequences of two or more different Cas13 guide RNAs, and / or, (a) The viral vector production components comprise one or more polynucleic acids collectively encoding the gene products of an AAV vector. Optionally, the viral vector components comprise the nucleic acid sequences of Rep52 or Rep40; Rep78 or Rep68; E2A; E4Orf6; VARNA; VP1; VP2; VP3; and AAP. Further optionally, the viral terminal repeats of the transferred polynucleic acid are AAV inverted tandem repeats, or, (b) The viral vector production components comprise one or more polynucleic acids collectively encoding the gene products of a lentiviral vector. Optionally, the viral vector components comprise the nucleic acid sequences of VSV-G, Gag-Pol and Rev. Further optionally, the viral terminal repeats of the transferred polynucleic acid are lentiviral long terminal repeats, and / or, At least one of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell, and / or, Each of the one or more heterologous polynucleic acids is stably integrated into the genome of the engineered cell, and / or, The engineered cell is derived from HEK293 cells, HeLa cells, BHK cells or Sf9 cells. **Claim 15** A method for reducing the expression of a payload molecule during viral vector production in the engineered cell according to claim 8 or 9, comprising (a) expressing shRNA or amiRNA during viral vector production, or, (b) expressing Cas13 and Cas13 guide RNA during viral vector production in the engineered cell according to claim 13 or 14.

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