JP-2022514761-A5 -

Dates

Publication Date

20221214

Application Date

20191219

Description

All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. The present invention may also be in the following embodiments. Item 1 An automated method for producing adeno-associated virus (AAV) viral vectors, (a) Introducing artificial mammalian AAV virus-producing cells into a completely sealed cell engineering system, wherein the artificial mammalian AAV virus-producing cells have their genome incorporated into them. i. Adenovirus helper genes, including E2A and E4Orf6 genes, under the control of the first desuppression promoter. ii. AAV genes, including the Rep and Cap genes, under the control of the second desuppression promoter. iii. Virus-associated non-coding RNA under the control of the third desuppression promoter, and iv. Introducing the suppression elements of the first, second, and third suppression release promoters, (b) Transduction of the mammalian AAV virus-producing cells with a vector encoding the target gene to produce transduction virus-producing cells, (c) Treating the mammalian AAV virus-producing cells with the binding partner of the inhibitory element, (d) Activating the first, second, and third suppression release promoters, (e) Proliferating the transdependence virus-producing cells and producing the AAV virus vector within the transdependence virus-producing cells, (f) Isolating the viral vector, (a) to (f) are methods performed in a closed automated process. Item 2 The method according to item 1, wherein the artificial mammalian AAV virus-producing cells are mammalian cell cultures. Item 3 The method according to item 2, wherein the mammalian cell culture is a suspension culture. Item 4 The method according to any one of items 1 to 3, wherein the artificial mammalian AAV virus-producing cells are Chinese hamster ovary (CHO) cells. Item 5 The method according to any one of items 1 to 3, wherein the artificial mammalian AAV virus-producing cells are human cells. Item 6 The method according to item 5, wherein the human cells are human embryonic kidney (HEK) cells. Item 7 The method according to any one of items 1 to 6, wherein each of the de-inhibition promoters comprises a functional promoter and two tetracycline operator sequences (TetO2 ) , and the repressive element is a tetracycline repressive protein. Item 8 The method according to item 7, wherein the treatment includes treatment with doxycycline. Item 9 The method according to any one of items 1 to 8, wherein the target gene is a gene to be treated. Item 10 The method according to any one of items 1 to 9, wherein the amount of AAV virus vector produced is at least about 10¹⁰ viral vectors. Item 11 The aforementioned closed-loop automated process, (a) Monitoring with one or more of the following: temperature sensor, pH sensor, glucose sensor, lactose sensor, oxygen sensor, carbon dioxide sensor, and optical density sensor, (b) The method according to any one of items 1 to 10, comprising automatically adjusting one or more of the following: temperature, pH level, glucose level, lactose level, oxygen level, carbon dioxide level, and optical density. Item 12 The method according to any one of items 1 to 11, wherein the transduction includes viral infection, electroporation, liposome transfection, or membrane disruption. Item 13 An automated method for producing an adeno-associated virus (AAV) viral vector, (a) Mammalian cells that stably express one or more nucleic acids encoding TetR and/or TetR-KRAB, i. A first nucleic acid encoding adenovirus helper genes, including the E2A gene, the E4Orf gene, and virus-associated non-coding RNA, under the control of a first desuppression promoter. ii. A second nucleic acid encoding the AAV genes, including the Rep and Cap genes, under the control of a second desuppression promoter, and iii. Transduction by a third nucleic acid encoding the target gene under the control of a third desuppression promoter, (b) Treating the mammalian cells with the binding partner of TetR, (c) Activating the first, second, and third suppression release promoters, (d) Proliferating the transduced cells and producing the AAV virus vector within the transduced cells, (e) Isolating the viral vector, (a) to (e) are methods performed in a closed automated process. Item 14 The method according to item 13, wherein the mammalian cells are mammalian cell cultures. Item 15 The method according to item 14, wherein the mammalian cell culture is a suspension culture. Item 16 The method according to any one of items 12 to 15, wherein the mammalian cells are Chinese hamster ovary (CHO) cells. Item 17 The method according to any one of items 12 to 15, wherein the mammalian cell is a human cell. Item 18 The method according to item 17, wherein the human cells are human embryonic kidney (HEK) cells. Item 19 The method according to any