November 25, 2020

The Niche

Knoepfler lab stem cell blog

Keep calm & CRISPR on: perspectives on report of human Cas9 immunity

The news that CRISPR-Cas9 gene editing in its current form may not work in a substantial fraction of people due to many of us having immunity to Cas9 came as a shock to many, but if you think about it, maybe it’s not so surprising. I don’t see it as the end of the world.

Motley Fool CRISPR stocks
Motley Fool Headline on CRISPR stocks
Keep calm & CRISPR on
CRISPR T-shirt

(preprint) from a group led by Matthew Porteus started this lively discussion a few days ago. The preprint is entitled, “Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans.” Some people are freaking out about this finding for a variety of reasons including for some investors their financial concerns (see the headline snapshot from the investing site Motley Fool above, for instance).

Everyone needs to take a deep breath.

If you think back to the fact that CRISPR-Cas9 as a toolbox for “gene editing” was developed from a natural bacterial defense system against viral infections, it’s logical that the bacterial Cas9 protein (the most commonly used nuclease by which CRISPR gene editing works via DNA repair) might be sometimes viewed as foreign by our immune systems as we grow up.

The new preprint reports that the two main sources of Cas9 protein so far from the bacteria Staph aureus (S. aureus) and Strep pyogenes (S. pyogenes), are likely going to be recognized as a sign of infection in some people. In fact, probably in many people. A lot of us are likely to be immune sensitive to these bacterial Cas9 proteins (reportedly, 79% to S. aureus Cas9 and 65% to S. pyogenes Cas9 in the limited number of donors examined in the preprint) because we’ll have antibodies against these Cas9 proteins.

CRISPR-Cas9 immunity
Adaptive Immunity to Cas9, challenge for CRISPR gene editing, Figure 1, Charlesworth, et al.

You can see in a pic from Figure 1 from the preprint that Western blots demonstrate endogenous human antibodies against Cas9 proteins often recognize clear protein bands. These human antibodies that make Western blot bands show up for Cas9 are in all likelihood present at a pretty decent concentration because so many of us have had and responded to S. aureus and S. pyogenes infections during our lifetimes and those bacteria express Cas9. Every time we are infected with a pathogen we stand a good chance of developing antibodies against unique proteins expressed by the pathogen and apparently Cas9 is fairly immunogenic.

Since many of us likely have Cas9 antibodies in our bloodstreams, if we were to receive CRISPR-Cas9 gene therapy our bodies may effectively inactive the CRISPR-Cas9 system via those antibodies. In addition, Porteus’ report (Charlesworth, et al.) found reactive T-cells as well in humans. Thus, not only might our antibodies inactivate CRISPR-Cas9, but also the resulting immune response could pose risks to patients getting CRISPR-Cas9 gene therapy. These data are so new though that it’s hard to know what will actually happen in patients who have Cas9 antibodies after getting CRISPR-Cas9 introduced into their systems. So, we know these antibodies are present in some people, but we don’t know the functional significance in a gene editing therapeutic setting. Another caveat for this paper is that the group of human donors was relatively small so in a wider, more genetically diverse population it is possible not such a high percentage of people are reactive to Cas9…or it could unfortunately go the other way and even more people may have antibodies. Functionally significant levels of Cas9 antibodies could be present yet undetectable so that’s another potential headache.

So what happens next?

A number of discussions are ongoing now about workarounds to this immunity hurdle, including some discuss in the piece from Antonio Regalado:

“New CRISPR systems are out there, just waiting to be discovered in bacteria that the human body has never seen—like those living in hydrothermal vents, say. Extracting cells from our bodies, treating them with CRISPR, and then putting them back might also work.”

These are good ideas.

Those of us with antibodies to S. aureus and/or S. pyogenes Cas9 may react with Cas proteins from other bacterial species. Still, it seems reasonably likely that other nucleases can be found that work well for gene editing but against which humans generally do not have antibodies. Importantly, transient use of Cas9 in patients’ cells in vitro followed by read ministration in vivo back into the patient could prove effective in getting good gene editing and at the same time avoiding immune responses. However, such an approach may not always be practical for gene editing of cell types other than those of the immune system. For example, you can’t easily remove brain precursor cells to gene edit in vitro without damaging the brain.

Other longer-standing issues are still out there too as some have pointed out such as human immune reactions to the viruses such as AAV sometimes used to deliver CRISPR-Cas9 systems. I don’t see any of these things as insurmountable though across the board. I believe some CRISPR-Cas9-based gene therapy will be proven safe and effective down the road.

Even more challenges and uncertainty await those who would use CRISPR in human embryos for proposed heritable prevention of genetic diseases as we can see exemplified by the complications and limbo status of the Mitalipov lab human embryo CRISPR paper from last year, the main conclusions of which were challenged by the Egli, et al. preprint (this is not even including societal and bioethical issues with heritable human gene editing). Note that I don’t see such ethical issues with CRISPR use for gene therapy.

The bottom line from this past week’s new finding on Cas9 antibodies and reactive T cells is that using CRISPR-Cas9 for applications such as gene therapy in humans is still almost certainly going to be workable, but in many cases it’ll be more complicated than hoped and other enzymes besides Cas9 might often be needed. The road ahead, as has been found to be true for so many transformative biomedical translational pipelines including for stem cells, is going to be tougher than imagined at first with more steps involved to maximize chances of success and lower risks for patients and the field. As a technology like CRISPR matures, the field needs to mature in our expectations and realize there will be big challenges along the way without panicking. Challenges are just the norm for science. In fact, usually the more exciting something is, the more hurdles we’ll run into along the way.

%d bloggers like this: