Compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells via viral delivery

Patent metadata

Field	Value
Publication number	US-11407994-B2
Application number	US-202217584298-A
Country	US
Kind code	B2
Filing date	Jan 25, 2022
Priority date	Apr 24, 2020
Publication date	Aug 9, 2022
Grant date	Aug 9, 2022

Abstract

This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth and mammalian cell transduction followed by nucleic acid-guided nuclease editing in live mammalian cells. The present compositions and methods entail viral delivery of an editing cassette to live mammalian cells such that the editing cassettes edit the cells and the edited cells continue to grow, preferably using a fully-automated end-to-end instrument to process the cells without human intervention to enhance cell processing uniformity and to maintain the integrity of the cell culture.

First claim

We claim:

1. A method of growing cells, passaging the cells, editing the cells via nucleic acid-guided nuclease editing, and detaching the cells in a bioreactor, comprising the steps of:
providing a bioreactor comprising:
a growth vessel comprising a tapered main body, a lid assembly comprising ports, at least one driving impeller, and an impeller shaft, wherein there is at least one liquid-in port; at least one liquid-out port; at least one gas-in port; at least one gas-out port; at least one rupture disc; and at least one sensor port; and wherein the lid assembly makes an air-tight fitting on the tapered main body; and
a bioreactor stand assembly comprising a frame, a stand main body disposed in the frame, wherein the stand main body accommodates the tapered main body of the growth vessel during operation, and wherein the stand main body comprises a heating element to heat the tapered main body;

providing cell growth medium, cells, and first microcarriers to the tapered main body;
allowing the cells to attach to the first microcarriers;
providing a viral vector to the tapered main body of the growth vessel, wherein each viral vector comprises an editing cassette and a selection marker, and wherein the viral vector is provided to the cells at a multiplicity of infection of less than one;
allowing the viral vectors to transduce the cells on the first microcarriers to produce transduced cells;
monitoring growth of the transduced cells on the first microcarriers;
selecting for transduced cells via the selection marker;
detaching the transduced cells from the first microcarriers;
allowing the first microcarriers to settle in the tapered main body of the growth vessel;
removing the first microcarriers from the tapered main body of the growth vessel;
adding reagent bundle microcarriers to the tapered main body of the growth vessel, wherein the reagent bundle microcarriers comprise a lipofection agent and a nuclease;
allowing the transduced cells to attach to and grow on the reagent bundle microcarriers;
providing conditions for the nuclease to transfect the transduced cells to produce transfected cells;
monitoring growth of the transfected cells on the reagent bundle microcarriers;
detaching the transfected cells from the reagent bundle microcarriers;
allowing the reagent bundle microcarriers to settle in the tapered main body of the growth vessel;
removing the detached transfected cells to a separate vessel.

2. The method of claim 1 , wherein the transduced and/or transfected cells are detached from the microcarriers via the at least one driving impeller.

3. The method of claim 1 , wherein the lid assembly further comprises a motor integration port for a motor to control the at least one driving impeller.

4. The method of claim 1 , wherein the bioreactor comprises a second driving impeller.

5. The method of claim 1 , wherein the at least one sensor port in the lid assembly is configured to accommodate a sensor to monitor capacitance of the cells and medium in the tapered main body of the growth vessel; a sensor to measure O 2 concentration of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure CO 2 of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure pH of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure lactate concentration of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure glucose concentration of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure biomass of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; or a sensor to measure optical density of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel.

6. The method of claim 5 , wherein there are at least two sensor ports in the lid assembly each configured to accommodate a sensor to monitor capacitance of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure O 2 concentration of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure CO 2 of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure pH of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure lactate concentration of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure glucose concentration of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; a sensor to measure biomass of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel; or a sensor to measure optical density of the transduced and/or transfected cells and medium in the tapered main body of the growth vessel.

7. The method of claim 1 , wherein the lid assembly further comprises a temperature probe.

8. The method of claim 1 , wherein the lid assembly further comprises a camera port.

9. The method of claim 1 , wherein the heating element of the stand main body comprises a heat jacket.

10. The method of claim 9 , wherein the heat jacket comprises LED lights.

11. The method of claim 9 , wherein the heat jacket comprises a camera port.

12. The method of claim 1 , wherein the at least one liquid-out port comprises a filter.

13. The method of claim 1 , wherein the tapered main body of the growth vessel accommodates cell culture volumes of 25 ml to 500 ml.

14. The method of claim 1 , wherein during cell growth revolutions per minute of the at least one driving impeller is approximately 40-80 rpm.

15. The method of claim 1 , wherein during cell detachment revolutions per minute of the at least one driving impeller is approximately 2700 rpm.

16. The method of claim 15 , wherein a chemical agent is added to the tapered main body of the growth vessel to aid in detaching the transduced and/or transfected cells.

17. The method of claim 16 , wherein the chemical agent is hemagglutinin, collagenase, dispase or trypsin.

18. The method of claim 1 , wherein the nuclease is provided as a protein.

19. The method of claim 1 , wherein the nuclease is provided as a nucleic acid coding sequence under control of a promoter.

20. The method of claim 19 , wherein the tapered main body is optically transparent in UV and IR ranges.

21. The method of claim 1 , wherein the tapered main body is optically transparent.

22. The method of claim 1 , wherein the stand frame is fabricated from stainless steel.

23. The method of claim 1 , wherein the bioreactor further comprises a liquid handling system wherein the liquid handling system comprises a manifold with one or more connections to the bioreactor.

24. The method of claim 1 , wherein the bioreactor further comprises a liquid handling system, wherein the liquid handling system comprises reagent receptacles individually connected to the growth module.

25. The method of claim 1 , wherein the cells are induced pluripotent stem cells (iPSCs).

26. The method of claim 1 , where

Assignees

• Inscripta Inc

Inventors

• Belgrader Phillip
• Siltanen Christian
• Watterson William
• Dura Burak
• Chabansky Bruce
• Stumbo David
• Smith Eric
• Bernate Jorge

Classifications

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N2750/14111

  Dependovirus, e.g. adenoassociated viruses · CPC title

• C12N2740/15011

  Lentivirus, not HIV, e.g. FIV, SIV · CPC title

• C12N2740/10011

  Retroviridae · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title