Stem cells are isolated and modified by using a lentiviral vector to add the ABCD1 gene. The stem cells in bone marrow are killed with chemotherapy, and replaced with the modified stem cells.
How often did cancer develop?
Hematologic cancer developed in 7 of 67 patients after the receipt of eli-cel
Yep, that's about what I expected. How long did it take?
myelodysplastic syndrome (MDS) with unilineage dysplasia in 2 patients at 14 and 26 months; MDS with excess blasts in 3 patients at 28, 42, and 92 months; MDS in 1 patient at 36 months; and acute myeloid leukemia (AML) in 1 patient at 57 months.
Makes sense. What does genetic analysis of the cancer cells indicate?
In the 6 patients with available data, predominant clones contained lentiviral vector insertions at multiple loci, including at either MECOM–EVI1 (MDS and EVI1 complex protein EVI1 [ecotropic virus integration site 1], in 5 patients) or PRDM16 (positive regulatory domain zinc finger protein 16, in 1 patient). Several patients had cytopenias, and most had vector insertions in multiple genes within the same clone; 6 of the 7 patients also had somatic mutations (KRAS, NRAS, WT1, CDKN2A or CDKN2B, or RUNX1), and 1 of the 7 patients had monosomy 7.
Multiple off-target insertions, different ones for different people. Yep.
Has this happened with gene therapy attempts before?
Yes, it's been a big problem with eg γ-retrovirus gene therapy trials. People hoped lentiviruses would be better because they tend to insert around actively transcribed genes, while γ-retroviruses tend to insert near transcription start sites.
Why don't we see high cancer rates after DNA viral infections?
We sort of do: Many cancers are strongly associated with viruses. But eli-cel is:
specifically done to stem cells, which are closer to cancer than usual
applied to cells away from the immune system, which can often detect infections and kill infected cells early
How about CRISPR instead of a viral vector?
Casgevy is a treatment that uses CRISPR on isolated cells. It has serious side effects, but not as bad as eli-cel. But a lot of people misunderstand what CRISPR actually does.
CRISPR is a technique that uses the Cas9 enzyme to cut double-stranded DNA according to added guide RNA. It doesn't insert DNA itself, but if DNA segments are added that are compatible with the cut section, they sometimes get inserted by homology-directed repair.
So, there are a few obvious issues here:
Cas9 has to be delivered to cells.
Sometimes Cas9 cuts the wrong spot; it depends on the sequences.
Also, DNA repair is different in different kinds of human cells, and CRISPR doesn't seem to work as well in non-stem cells.
Currently, physical methods (microinjection technology, electroporation, and HTVI) are commonly used for delivering CRISPR/Cas9. But those aren't very practical in live animals, and also have large effects on cells.
How about mRNA delivery of CRISPR, then?
Yes, delivering mRNA that codes for Cas9 can work. It might be a better approach for medicine.
That will still probably be limited to isolated cells in general. In live animals, you run into a lot of problems with immune system reactions to Cas9; when that's the desired effect you have mRNA vaccines, which are rather well-known now.
After treatment, any proteins that weren't already being produced can also cause an immune reaction. Eli-cel hasn't had as many immune system rejections as transplants, but it's still been a problem. It doesn't work as well in people with the ABCD1 gene fully deleted instead of nonfunctional, probably because the fixed protein is similar enough to the (already-produced) flawed version to not have an immune response.
If Cas9 doesn't insert genes, how about bridge RNAs with IS110, that fancy RNA-guided DNA editing technique that was in the news?
That currently only works for bacteria. The guy who discovered it wants to use AI to modify that enzyme so it works in humans, but I think that's probably not usable for eukaryotes. It seems useful for making GM microbes, but plasmid synthesis is already easier than CRISPR.
OK then, how about editing a virus so whatever inserts viral DNA is more selective?
Ah, a modified retroviral integrase? A selective one won't evolve naturally because the success rate of inserting something somewhere will be lower, but the existence of eg (Cas9 and IS110 and homology-directed repair) shows such an enzyme should be possible. One of my friends has actually worked on this a bit, but they're sort of holding back because of concerns about bioweapons. (They're smarter than me and I'm not qualified to second-guess their concerns.)
Even if the integrase of a virus can't be made sequence-specific, there could still be benefits to modifying its insertion tendencies. If a lentivirus is better than a γ-retrovirus in terms of where it tends to insert DNA, then maybe you can do better than either.
Here's a new paper on cancer developing in patients after receiving gene therapy with eli-cel, brand name Skysona.
What is eli-cel?
Stem cells are isolated and modified by using a lentiviral vector to add the ABCD1 gene. The stem cells in bone marrow are killed with chemotherapy, and replaced with the modified stem cells.
How often did cancer develop?
Yep, that's about what I expected. How long did it take?
Makes sense. What does genetic analysis of the cancer cells indicate?
Multiple off-target insertions, different ones for different people. Yep.
Has this happened with gene therapy attempts before?
Yes, it's been a big problem with eg γ-retrovirus gene therapy trials. People hoped lentiviruses would be better because they tend to insert around actively transcribed genes, while γ-retroviruses tend to insert near transcription start sites.
Why don't we see high cancer rates after DNA viral infections?
We sort of do: Many cancers are strongly associated with viruses. But eli-cel is:
How about CRISPR instead of a viral vector?
Casgevy is a treatment that uses CRISPR on isolated cells. It has serious side effects, but not as bad as eli-cel. But a lot of people misunderstand what CRISPR actually does.
CRISPR is a technique that uses the Cas9 enzyme to cut double-stranded DNA according to added guide RNA. It doesn't insert DNA itself, but if DNA segments are added that are compatible with the cut section, they sometimes get inserted by homology-directed repair.
So, there are a few obvious issues here:
Also, DNA repair is different in different kinds of human cells, and CRISPR doesn't seem to work as well in non-stem cells.
Currently, physical methods (microinjection technology, electroporation, and HTVI) are commonly used for delivering CRISPR/Cas9. But those aren't very practical in live animals, and also have large effects on cells.
How about mRNA delivery of CRISPR, then?
Yes, delivering mRNA that codes for Cas9 can work. It might be a better approach for medicine.
That will still probably be limited to isolated cells in general. In live animals, you run into a lot of problems with immune system reactions to Cas9; when that's the desired effect you have mRNA vaccines, which are rather well-known now.
After treatment, any proteins that weren't already being produced can also cause an immune reaction. Eli-cel hasn't had as many immune system rejections as transplants, but it's still been a problem. It doesn't work as well in people with the ABCD1 gene fully deleted instead of nonfunctional, probably because the fixed protein is similar enough to the (already-produced) flawed version to not have an immune response.
If Cas9 doesn't insert genes, how about bridge RNAs with IS110, that fancy RNA-guided DNA editing technique that was in the news?
That currently only works for bacteria. The guy who discovered it wants to use AI to modify that enzyme so it works in humans, but I think that's probably not usable for eukaryotes. It seems useful for making GM microbes, but plasmid synthesis is already easier than CRISPR.
OK then, how about editing a virus so whatever inserts viral DNA is more selective?
Ah, a modified retroviral integrase? A selective one won't evolve naturally because the success rate of inserting something somewhere will be lower, but the existence of eg (Cas9 and IS110 and homology-directed repair) shows such an enzyme should be possible. One of my friends has actually worked on this a bit, but they're sort of holding back because of concerns about bioweapons. (They're smarter than me and I'm not qualified to second-guess their concerns.)
Even if the integrase of a virus can't be made sequence-specific, there could still be benefits to modifying its insertion tendencies. If a lentivirus is better than a γ-retrovirus in terms of where it tends to insert DNA, then maybe you can do better than either.