* Prepare a DNA plasmid with the Cas9 gene, guide RNA for the desired genetic modification, and an antibiotic resistance gene.
* Electroporate the plasmid into the harvested stem cells.
Grow the electroporated stem cells in antibiotic-containing nutrient media. Only cells with the plasmid (and thus antibiotic resistance) survive.
* Expand and freeze the genetically modified cells.
* Administer chemotherapy to the patient to eliminate defective bone marrow stem cells.
*
Inject the modified stem cells back into the patient, where they repopulate the bone marrow with the CRISPR edits, aiming to correct the genetic mutation.
This process isn't new but one of the biggest challenges is propagating genetic modifications to all effected cells in the body. This is why it's much easier to GMO an egg / sperm because once the change is made there, it's replicated in every new cell thereafter.
Other techniques utilize harmless viruses to transfect genetic modifications to the body, but this has other trade-offs. mRNA vaccines don't propagate to every cell, but the cells which do successfully transcribe the mRNA are able to generate enough of the target protein that the body can recognize it and develop an immunity to it. Eventually, the modified cells will die and no cells will be left to produce the mRNA vaccine protein.
Do you know why they're having the marrow synthesise fetal hemoglobin versus hemoglobin A(2)? (Is it simply because HbF is one molecule while HbA and HbA2 are two?)
Fetal hemoglobin has a lot of biochemical advantages for fighting sickle-cell disease on its own so this is leveraged in the CRISPR solution - e.g. create more of the cells that inhibit the disease in the first place.
AAV or adeno-associated virus is a delivery method for getting cas9 mRNA (the code that says, make cas9 protein and do gene editing). Zinc finger nucleases are a similar class of dna editing proteins.
In this specific experiment they chose to transfect cells with plasmid directly rather than transduce with virus.
ZFNs are difficult and slow to engineer. There are certainly tradeoffs but the fact that almost the entire industry is using CRISPR-based approaches tells you where things lie on balance
* Harvest stem cells from the patient.
* Prepare a DNA plasmid with the Cas9 gene, guide RNA for the desired genetic modification, and an antibiotic resistance gene.
* Electroporate the plasmid into the harvested stem cells. Grow the electroporated stem cells in antibiotic-containing nutrient media. Only cells with the plasmid (and thus antibiotic resistance) survive.
* Expand and freeze the genetically modified cells.
* Administer chemotherapy to the patient to eliminate defective bone marrow stem cells.
* Inject the modified stem cells back into the patient, where they repopulate the bone marrow with the CRISPR edits, aiming to correct the genetic mutation.
This process isn't new but one of the biggest challenges is propagating genetic modifications to all effected cells in the body. This is why it's much easier to GMO an egg / sperm because once the change is made there, it's replicated in every new cell thereafter.
Other techniques utilize harmless viruses to transfect genetic modifications to the body, but this has other trade-offs. mRNA vaccines don't propagate to every cell, but the cells which do successfully transcribe the mRNA are able to generate enough of the target protein that the body can recognize it and develop an immunity to it. Eventually, the modified cells will die and no cells will be left to produce the mRNA vaccine protein.