Combining these two systems allows Memphis Meats to use DOX and E2 to control behavior of the O2K cell line. They start the cells in a DOX-containing medium to induce proliferation, then later switch to an E2-containing medium to induce myogenesis. They also include the GSK3B inhibitor CHIR99021 in both media to prevent cell death while transitioning from the proliferation to the differentiation phase, and the DNA methylation inhibitor 5-Azacytidine (5AC) in the differentiation media to help terminal differentiation.

Claims [3]: While the body of the application covers the version of the double-switch system involving the O2K cell line, and the cell control program using DOX, E2, CHIR99021 and 5AC, the claims are much broader, and protect the entire double-switch strategy. This includes any process which genetically modifies a self-renewing cell line so that it’s possible to exogenously induce proliferation, and separately exogenously induce myogenesis. The application has not been granted yet, so the claims may change if the patent office determines they are too broad.

I expect this to be an appealing strategy for companies using GMOs since it for allows control over cell behavior while lowering the cost of the media by eliminating expensive growth factors. This could be useful for scaling up a process, or as a research tool for working with myogenic cell lines.

Methods for extending the replicative capacity of somatic cells during an ex vivo cultivation process (2016, Patent Pending)

This patent application describes a novel way to immortalize a cell line using genetic engineering. It builds on an existing method of immortalizing myogenic cell lines by overexpressing telomerase reverse transcriptase (TERT) and cyclin-dependent kinase 4 (CDK4) [4]. Memphis Meats’ innovation is that instead of overexpressing CDK4, they use CRISPR to knock out a protein that naturally inhibits it. This allows them to immortalize cell lines without having to ectopically express CDK4.

The CDK4-inhibiting genes that Memphis Meats targets are CDKN2A for chicken cell lines, and CDKN2B for cow cell lines. Memphis Meats uses CRISPR to knock them out by making small insertions at the locus of each gene, causing frameshift mutations. The tumor-suppressing proteins p15 and p16 are then no longer expressed. Since these proteins naturally inhibit CDK4, their absence has the same end effect as ectopic expression of CDK4.

It seems like the main benefit of this method is that it uses gene editing over genetic modification—instead of adding an entirely new gene to ectopically express CDK4, it makes a small modification to a gene that already exists in the cell. This could be favorable from a regulation standpoint, given existing precedent in plants. There’s still a lot of uncertainty over how cell-based meat will be regulated, but crops that are merely gene edited are not considered GMO (unless you’re in Europe [5]).

Claims: This patent application protects any method of immortalizing a cell line for meat that involves ectopically expressing TERT, and using genetic engineering to knock out any cyclin-dependent kinase inhibitor (e.g. CDKN2A and CDKN2B). Again, the claims are much broader than the technology discussed in the patent application. This patent application is relatively new, meaning that the claims might get narrower during the review process.

Interestingly, the independent claim of the application specifies that the TERT must be ectopically expressed. This means that this patent application only protects processes that would yield GMO meat, even though the main benefit of the application is that it uses gene editing instead of genetic modification. If you increase TERT levels in some other non-GM way (e.g. serial passaging a cell line and selecting for clones with high naturally TERT expression), you could create a non-GMO immortalized muscle cell line that wasn’t protected by this application. There could also be a different benefit to this method that’s not explicitly discussed in the patent application.

Just