Safety: CRISPR cause virus vector to integrate at high level. Interview: Bence György

- Virus vectors integrating into host DNA sounds very bad as in risk of causing cancer, how concerned should we be, is this going to end AAV's as vectors for CRISPR gene therapy?

No no no, we are not saying we can't use AAV for CRISPR delivery. In fact, our data suggests that at least in the rodent system expression of Cas9 and gRNAs does not increase AAV integration. AAV vectors are known to integrate at a low rate (~0.1%) of incoming vector at DNA breaks. Since CRISPR makes DNA breaks, in hindsight, it isn’t surprising that AAV also integrates into these cut sites. The important observation that we made is that this basal level of integration is not enhanced with CRISPR, and that the very specific cutting with well-designed guides just provides AAV with another potential integration site. At this point we have to characterize the AAV CRISPR system fully and make sure we know that integration is happening and detect it with specific protocols.

AAV integration is very common

- ok, lets take a few steps back, can you explain a bit about the background for the paper?

Yes. We previously published a Nature Medicine paper on a dominant form of hearing loss. We used AAV CRISPR to make a doublestranded break and knock out the function of dominant deafness gene, Tmc1, in inner-ear hair cells.

We had mice that were completely deaf and after AAV CRISPR injection they were normal hearing up to one year after. It was really astonishingly.

- yes?

Since AAV already integrates, and CRISPR is only adding one additional site, it does not obviously impact safety concerns over what is already occuring with AAV vectors in the first place.

However, if poor guides (non-specific) guides were designed, one could imagine more off-target cuts would be made and this could increase AAV integration over basal levels. So it is important to use the most efficient Cas9/gRNA system for your target.

Additionally, one should consider where the cut site is in the context of other genes/oncogenes. Integration of the CMV-promoter driven Cas9 could potentially activate genes you don’t want to activate. However, this has not been reported in any clinical trials with AAV vectors which already integrate on their own.

AAV normally safe non-integrating vector

- Ok, before continuing maybe you can explain a bit why you use AAV vector, what makes it a good vector?

Yes. First of all the AAV or Adeno-Associated Virus is a very safe non-pathogenic virus that has been accepted by the FDA as a therapy for two diseases so far, retinal therapy for blindness, and spinal muscular atrophy to treat a horrific disease in infants. It is very effective and currently there is no better delivery tool for nondividing tissue like brain, muscle, eye or ear.

- but being in non-dividing cells also means it will stay and express the CRISPR Cas9 for a long time?

Correct, this is a concern. In dogs AAV encoded transgenes have been shown that expression can be there for a decade. So this is a concern because it is a long term expression and there is a risk because something might happen even a decade later (like immunogenicity to Cas9 or off target cutting).

Of course there are many questions. You have to ask, what will happen? Formation of a tumor or may be an immune reaction at some point or degeneration of the target cells?

Basically we now know the integration is happening already with AAV vectors and we have to look at the safety aspects and analyze this specifically. From our studies we never saw hard outcomes like increase in mortality, cancer formation or genotoxicity.