My lab does research specifically on in vitro gene editing of T-cells, mostly via Lentivirus and electroporation, and I can tell you that this problem is HARD.
Are you doing traditional gene therapy or CRISPR-based editing?
If the former, I’d guess you’re using Lentivirus because you want genome integration?
Even in-vitro, depending on the target cell type and the amount/ it is very difficult to get transduction efficiencies higher than 70%, and that is with the help of chemicals like Polybrene, which significantly increases viral uptake and is not an option for in-vivo editing.
Does this refer to the proportion of the remaining cells which had successful edits / integration of donor gene? Or the number that were transfected at all (in which case how is that measured)?
Essentially, in order to make this work for in-vivo gene editing of an entire organ (particularly the brain), you need your transduction efficiency to be at least 2-3 orders of magnitude higher than the current technologies allow on their own just to make up for the lack of polybrene/retronectin in order to hit your target 50%.
This study achieved up to 59% base editing efficiency in mouse cortical tissue, while this one achieved up to 42% prime editing efficiency (both using a dual AAV vector). These contributed to our initial optimism that the delivery problem wasn’t completely out of reach. I’m curious what you think of these results, maybe there’s some weird caveat I’m not understanding.
The short answer is that they are, but they are doing it in much smaller steps. Rather than going straight for the holy grail of editing an organ as large and complex as the brain, they are starting with cell types and organs that are much easier to make edits to.
This is my belief as well—though the dearth of results on multiplex editing in the literature is strange. E.g. why has no one tried making 100 simultaneous edits at different target sequences? Maybe it’s obvious to the experts that the efficiency would be to low to bother with?
Really interesting, thanks for commenting.
Are you doing traditional gene therapy or CRISPR-based editing?
If the former, I’d guess you’re using Lentivirus because you want genome integration?
If the latter, why not use Lipofectamine?
How do you use electroporation?
Does this refer to the proportion of the remaining cells which had successful edits / integration of donor gene? Or the number that were transfected at all (in which case how is that measured)?
This study achieved up to 59% base editing efficiency in mouse cortical tissue, while this one achieved up to 42% prime editing efficiency (both using a dual AAV vector). These contributed to our initial optimism that the delivery problem wasn’t completely out of reach. I’m curious what you think of these results, maybe there’s some weird caveat I’m not understanding.
This is my belief as well—though the dearth of results on multiplex editing in the literature is strange. E.g. why has no one tried making 100 simultaneous edits at different target sequences? Maybe it’s obvious to the experts that the efficiency would be to low to bother with?