>"The researchers removed immune cells from the recipient’s blood and then disabled a gene in them using CRISPR–Cas9, which combines a DNA-cutting enzyme with a molecular guide that can be programmed to tell the enzyme precisely where to cut. The disabled gene codes for the protein PD-1, which normally puts the brakes on a cell’s immune response: cancers take advantage of that function to proliferate.
Lu’s team then cultured the edited cells, increasing their number, and injected them back into the patient, who has metastatic non-small-cell lung cancer. The hope is that, without PD-1, the edited cells will attack and defeat the cancer."
Or maybe it was this:
>"The researchers removed immune cells from the recipient’s blood and then selectively killed most cells containing a certain sequence using CRISPR–Cas9, which combines a DNA-cutting enzyme with a molecular guide that can be programmed to tell the enzyme precisely where to cut. The targeted gene codes for the protein PD-1, which normally puts the brakes on a cell’s immune response: cancers take advantage of that function to proliferate.
Lu’s team then cultured the surviving cells, increasing their number, and injected them back into the patient, who has metastatic non-small-cell lung cancer. The hope is that, without PD-1, the selected-for cell population will attack and defeat the cancer."
Since there is no paper (only press release) we can't say much more about which explanation is most plausible in this case.
Luckily the article linked to the clinical trials.gov record for this trial [1] which suggests that the first approach (editing vs selection) is being used.
That seems to be the favorite interpretation. We need to see what data they present that favors it (ie % survival of the treated cells, number of initial cells, % mutants at that location detected in control cells). Also, this is kind of weird, because they say it is non-randomized:
Study Design:
Allocation: Non-Randomized
Endpoint Classification: Safety Study
Intervention Model: Parallel Assignment
Masking: Open Label
Primary Purpose: Treatment
But later it says:
Progression free survival - PFS [ Time Frame: From date of randomization until
the date of first documented progression or date of death from any cause, whichever
came first, assessed up to average 10 months ] [ Designated as safety issue: No ]
Overall Survival - OS [ Time Frame: The time from randomization to death from any
cause, assessed up to 2 years ] [ Designated as safety issue: No ]
So is the treatment randomized or not? The info on that site may not be reliable... maybe it makes sense somehow though.
I would be surprised if this safety trial were randomized with respect to the CRISPR manipulation. I agree with your assessment of needing more data to believe that the gene editing manipulation was successful.
I suspect the randomization relates to the allocation to the experimental arms as described on the reporting page:
This is a dose-escalation study of ex-vivo knocked-out,
expanded, and selected PD-1 knockout-T cells from
autologous origin. Patients are assigned to 1 of 3
treatment groups to determine the maximal tolerant dose
>"CRISPR is used to mess with a gene, not kill a population of cells."
Look it up, in every paper where they report on that aspect, the vast majority of the cells die. Also, they detect small numbers of mutants in the control (non-"modified") cells.
I haven't read a ton of CRISPR/CAS papers, but I have read the seminal ones, and didn't see that. Mutations in unmodified cells are completely expected, of course, but not mutations in line with where CRISPR is being targeted—I'd hardly believe that to be the case. It looks like the cell perforation (getting CRISPR/CAS into the cells) can certainly affect cell viability, but those numbers have been steadily, rapidly improving. Quick search shows that in 2015, a study found 80% viability post perforation and CRISPR/CAS application (http://advances.sciencemag.org/content/1/7/e1500454.full).
Looking at that paper, the 80% viability doesn't refer to after CRISPR/Cas9 had time to do the selection, it refers to passage through their chip and puromycin selection (which just indicates the plasmid got in the cell):
"Plasmids encoding Cas9 and sgRNA targeting phosphatase and tensin homolog (Pten) (fig. S6A) were delivered into MCF7 cells, followed by culture for 48 hours and puromycin selection. More than 80% of the cells survived the selection process, indicating the high delivery efficiency of our method.
[...]
Similar to Pten knockout, more than 80% of 53BP1 knockout cells survived the
selection process."
You have to look at figure 5B, where the caption reads:
"Cells (5 × 10^4) from (A) were seeded in 60-mm dishes in complete medium and cultured for 7 days. Cells were trypsinized and collected for cell count in a Countess II FL Automated Cell Counter (Life Technologies) daily for 7 days."
So they started with 5 x 10^4 cells but if you look at the first day after being in culture, it is much less than that. I can't tell how low from the chart which shows it as basically indistinguishable from zero. They do not seem to report those values anywhere.
Also, you can look at figure S5 and see that they saw GFP get knocked out in >2% of control cells (Cas9 only). They don't do a similar quantification for the Pten, but if they had, I am pretty sure some low percentage of cells would be shown as already knocked out for Pten.
Lu’s team then cultured the edited cells, increasing their number, and injected them back into the patient, who has metastatic non-small-cell lung cancer. The hope is that, without PD-1, the edited cells will attack and defeat the cancer."
Or maybe it was this:
>"The researchers removed immune cells from the recipient’s blood and then selectively killed most cells containing a certain sequence using CRISPR–Cas9, which combines a DNA-cutting enzyme with a molecular guide that can be programmed to tell the enzyme precisely where to cut. The targeted gene codes for the protein PD-1, which normally puts the brakes on a cell’s immune response: cancers take advantage of that function to proliferate.
Lu’s team then cultured the surviving cells, increasing their number, and injected them back into the patient, who has metastatic non-small-cell lung cancer. The hope is that, without PD-1, the selected-for cell population will attack and defeat the cancer."
Since there is no paper (only press release) we can't say much more about which explanation is most plausible in this case.