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Comment by eganist | Hacker News Reader

Way out of my depth here, but I'm guessing the main hurdles to turning this into a drug are encapsulation in a way that survives injection (or better yet, ingestion) as well as either targeting of infected cells or at least ensuring that even if it touches every cell, it only affects HIV DNA?

Basically, what's needed to commercialize this?

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There's a sickle cell therapy that uses CRISPR. I don't know the specifics but if one has been successful then a second can be too. I'm looking forward to getting more information in the future. Cell culture vs cure is a long road but at least there's hope. The big drawback is that these therapies are super expensive but time and experience in the past have proven that the costs can be lowered.

"Sickle Cell Disease Approvals Include First CRISPR Gene Editing Therapy"

https://jamanetwork.com/journals/jama/article-abstract/28137....

Yup Casgevy & Lyfgenia! These sickle cell (and now also approved for beta thalassemia) CRISPR therapies basically work in the following way:

- Blood stem cells are removed from the patient and the CRISPR Cas9 protein outside of the body is injected to cut the gene responsible for suppressing fetal hemoglobin production (even people with sickle cell have healthy fetal hemoglobin, their adult hemoglobin gene is what causes the deformed red blood cells)

- Chemotherapy is used to kill all living bone marrow and remove all previous unedited stem cells.

- New edited stem cells are inserted, and patient recovers with new blood production being of healthy red blood cells.

I'd say a huge step forward was FDA and EMA approval, but figuring out a way to remove previous unedited stem cells with chemotherapy would be a step change in the patient experience.

It's both extremely sci-fi and incredibly terrifying that one of the steps for a cure is to quote unquote kill all living bone marrow. Modern medicine is fascinating in how advanced it can be.

Yeah it's a spectacular transplant procedure, though it's really not that modern medicine, it has been developed since the 50s! The step where you irradiate or apply chemo to kill off the existing bone marrow had to be done at first because the patients had leukemia so you had to do this anyway. An interesting thing is that if there are still cells left in you, they will be wiped clean by the new transplanted immune system in a so called graft-vs-host response that also sounds like a horror-movie concept :)

And yet, measles are back.

not sure if you need to kill absolutely all living bone marrow. Unlike cancer having 5% sickle cell red blood cells is probably fine. That makes the chemo probably way less bad than what you would need for cancer.

"- Chemotherapy is used to kill all living bone marrow and remove all previous unedited stem cells.

- New edited stem cells are inserted, and patient recovers with new blood production being of healthy red blood cells."

It's very likely HIV could be cured similarly. I believe all the people who have been cured so far are bone marrow transplant recipients in which the marrow had a specific gene or genes.

It's very different, you only have to fix some of the bone marrow to help someone with sickle-cell, but you have to eliminate all reservoirs of HIV to cure AIDS.

I was under the understanding that HIV attacks only immune cells, white blood cell, only. All the issues that eventually kill are due to the body not being able to defend itself. So it seems like a cure would be very much similar to a sickle cell cure. Is that not the case?

A good video explanation of that too via Sci Show. It's quite complex; chemo, lab work and takes a long time and not always successful but the majority of the time it is successful.

https://www.youtube.com/watch?v=uHWD8RSw4As

Generally speaking:

- Delivery: getting the mRNA and guide RNA into the target cells

- Expression: ensuring the target cell expresses the mRNA, thus making the protein

- On Target Editing Efficiency: ensuring the intended edit happens at a high rate

- Off Target Editing: ensuring other edits in other locations do not occur

crispr also makes mistakes. The kind that lead to cancer, disability and death. It’s not a magic wand. Being able to do something in cell culture is interesting but very far away from a therapy.

How do Casgevy and Lyfgenia[1] avoid these dangerous mistakes? Is it something that can be generalized to other therapies, or does it need to be recreated for each "edit" that we want to turn into a therapy?

1. https://www.fda.gov/news-events/press-announcements/fda-appr...

According to your link, both of those are applied to extracted stem cells which are then reintroduced. The ability to perform clonal expansion and DNA sequencing makes it possible that screening for off-target edits could be performed. Though I have no idea if it's actually done.

Your point is broadly correct (not sure about disability though), but I think it overstates the danger of what will happen in practice. Yes, if you picked a random target and blasted away, you probably wouldn't have a good time. But anything that even approaches clinical trials is going to get substantial engineering put into it to minimize (and characterize) all of the off-target loci. If there's meaningful editing near an oncogene then that's going to be a deal breaker for a particular guide. When the FDA was discussing the Casgevy application, they went into remarkable detail of how Vertex had measured the off-targets - the regulators really, really don't want to approve anything risky.

Frankly just getting it to work at all is the real hurdle in this case.

> If there's meaningful editing near an oncogene then that's going to be a deal breaker for a particular guide.

At this point do we know every gene that had the potential to cause cancer?

We roughly know the space of possible mutations in the human genomes because we have so many sequenced genomes now: if we don't spot a mutation it's probably not good when it happens, survivorship bias.

The problem with CRISPR is that we cannot control where the off-target effects happen, we can currently only optimise the guiding RNA and the Cas enzyme to have as little off-target effects as possible (but not 0, yet). It would be cool to engineer guiding RNAs that bind in those high mutation-rate areas when they have off-target effects, stuff can mutate there and nothing will happen (probably).

> Frankly just getting it to work at all is the real hurdle in this case

This being the point. Many diseases have been cured dozens of times over in a tissue sample in a lab that never make it to actual therapies because the hurdle is elsewhere. The risks with crispr are as stated, especially flooding your entire body with it to target a virus.

> Your point is broadly correct (not sure about disability though), but I think it overstates the danger of what will happen in practice

https://www.nature.com/articles/nrc1122

One thing I was confused by: why did Vertex choose to reactivate HbF instead of attempting to correct the mutation that causes sickle cell disease in the first place?

CRISPR, in the form used by Vertex, is not capable of directly repairing an existing gene. In the case of sickle cell this means directly changing the mutated nucleotide in the HbA gene. CRISPR is very capable of cutting the genome at precise locations. These cuts lead to lossy repair pathways that introduce mutations or deletions that disable the gene at the spot that CRISPR cut. So, the best you can hope for is that the CRISPR cut leads to a loss of function. It's possible to use CRISPR to introduce new sequences into the DNA, by introducing a new DNA sequence alongside the CRISPR proteins, then hoping that DNA repair "accidentally" uses the genetic sequence you put in to repair the break in the DNA. This is even less efficient than just cutting the DNA, and it would not fix the mutated HbA, so it's not really therapeutically relevant for sickle cell.

There are more recent techniques, notably prime editing, that use a modified version of the CRISPR system that can introduce changes to single bases (nucleotides) in the genome. These have some promise of directly fixing diseases caused by single mutations, but there are hurdles in terms of efficiently delivering the prime editor to the right tissues as well as efficiency of the actual repair.

So how is Vertex activating HbF? Did they find some bases to remove that cause the gene to be expressed more than usual?

They’re snipping another gene (BCL11A) that silences, or turns off, HbF. Turning off BCL11A then allows HbF to be expressed

Does this means that in order to productionize this into a drug, one would need to basically engineer a biological virus?

https://en.wikipedia.org/wiki/Viral_vector

or mRNA

CRISPR is pretty complicated - I'd say it's not clear whether mRNA of such length could stably produce enough enzyme in enough places. It's great when you need an antigen to be presented, but not so much when you need an entire restriction enzyme system.

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