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-11268088-B2
Application number	US-202117239538-A
Country	US
Kind code	B2
Filing date	Apr 23, 2021
Priority date	Apr 24, 2020
Publication date	Mar 8, 2022
Grant date	Mar 8, 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 transducing, transfecting, and performing nucleic acid-guided nuclease editing in mammalian cells in an automated closed editing instrument comprising the steps of:
providing an automated closed cell editing instrument comprising a growth module and a liquid handling system;
transferring cell growth medium, first microcarriers and mammalian cells to the growth module via the liquid handling system;
growing the mammalian cells on first microcarriers in the growth module;
synthesizing and amplifying a library of editing cassettes off-instrument, wherein each editing cassette comprises a different gRNA and donor DNA pair;
inserting editing cassettes into a viral backbone and producing viral vectors off-instrument;
delivering the viral vectors to the mammalian cells in the growth module at a multiplicity of infection (MOI) such that each mammalian cell receives one viral vector or no viral vector;
providing conditions to allow the viral vector to integrate into the mammalian cell DNA;
after integration, growing the detached mammalian cells;
enriching for mammalian cells with comprising an integrated viral vector;
detaching the enriched mammalian cells from the first microcarriers;
synthesizing off instrument second microcarriers comprising lipfectamine and a nucleic acid-guided nuclease or nuclease fusion or a coding sequence for a nucleic acid-guided nuclease or nuclease fusion;
delivering the second microcarriers comprising lipofectamine and a nucleic acid-guided nuclease or nuclease fusion or a coding sequence for a nucleic acid-guided nuclease or nuclease fusion to the detached mammalian cells in the growth module via the liquid handling system;
allowing the delivered nucleic acid-guided nuclease or nuclease fusion to transfect the enriched mammalian cells; and
providing conditions to allow editing to take place in the mammalian cells.

2. The method of clam 1 , wherein the growth module is a bioreactor.

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

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

5. The method of claim 1 , wherein the mammalian cells are primary cells.

6. The method of claim 1 , wherein the first and/or second microcarriers are fabricated from natural organic materials, biocompatible synthetic polymers, or inorganic materials.

7. The method of claim 6 , wherein the first and/or second microcarriers are fabricated from polystyrene.

8. The method of claim 6 , wherein the first and/or second microcarriers are fabricated from a polyacrylate.

9. The method of claim 6 , wherein the first and/or second microcarriers are coated with laminin.

10. The method of claim 9 , wherein the first and/or second microcarriers are coated with laminin L-521.

11. The method of claim 1 , wherein the first and/or second microcarriers range in size from 30-1200 microns in diameter.

12. The method of claim 11 , wherein the first and/or second microcarriers range in size from 50-150 microns in diameter.

13. The method of claim 1 , wherein after the enriching and second delivering step, the mammalian cells are detached from the second microcarriers, the medium is exchanged and fresh microcarriers are added to the growth module.

14. The method of claim 13 , wherein the growth module is a bioreactor with at least one impeller and the mammalian cells are detached from the first and/or second microcarriers by increasing revolutions per minute of the at least one impeller.

15. The method of claim 13 , wherein the growth module is a bioreactor with at least two impellers and the mammalian cells are detached from the first and/or second microcarriers by increasing revolutions per minute of the at least two impellers.

16. The method of claim 1 , wherein the viral vector is a lentiviral vector.

17. The method of claim 1 , wherein the viral vector is an adeno-associated virus vector.

18. The method of claim 17 , wherein the viral vector is delivered to the cells on the first microcarriers at an MOI of <0.1-0.5.

19. The method of claim 1 , wherein the viral vector is an oncoretrovirus vector.

20. The method of claim 18 , wherein the viral vector is delivered to the cells on the first microcarriers at an MOI of <0.1-0.3.

21. The method of claim 1 , wherein the viral vector is delivered to the cells on the first microcarriers at an MOI of <0.05-1.0.

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