At the end of 2018, for the first time in history, the two Chinese twins Lulu and Nana were born with their genome modified by the CRISPR / Cas9 system. This outraged the entire scientific community and ask us about the risks of therapies targeting Human genome. The Cas9 nuclease is often compared to DNA scissors, allowing a very precise cut into any gene. CRISPR are the hands that position these scissors at the desired site of the genome. This system promises to be able to repair any defective gene, thus appearing as the solution for many genetic diseases. It is a valuable tool as well for many therapeutic approaches using genome engineering.

The off target effect is due to a lack of specificity from Cas 9 protein

Talking about gene editing, the term "off-target" is used to describe the fact that the Cas9 protein is not 100% accurate and can also cut the genome at unwanted sites. "Off-target" also refers to all the unwanted effects that the CRISPR / Cas9 tool can generate. Indeed, the mechanisms of DNA repair engaged after the cut produced by the Cas9 scissors can create unexpected mutations. Thus, the impact of using the CRISPR / Cas9 system is often underestimated. Today, we are only at the beginning of clinical research and we are not yet fully managing the risks of CRISPR / Cas9 therapy in humans. However, there are already a few solutions that can be worked out.

The first technical challenges are about targeting accuracy

Inactivating a defective gene, replacing it or even "only" fixing it by a slight modification implies a perfect precision. Hundreds of genes succeed each other on our genome, it is therefore crucial to target the desired gene and not the one beside. In addition, a gene is made up of different bricks, all of which play a different role, and again, we must target the good one. Finally, each gene is present in each of our cells, the CRISPR system must also be effective in a maximum of cells to be efficient. To summarize, the perfect tool must be effective and precise but, most importantly, manageable.

The natural mechanism of DNA repair is not perfect neither

Understanding the natural mechanisms of cell repair is a second challenge. The cellular repair system helps to maintain the organism integrity by constantly monitoring our DNA. Several repair systems exist within cells to ensure that any damaged DNA fragment does not persist. When the genetic scissors of the CRISPR system cut the DNA, the cell will immediately try to repair the break in the gene. The repair may consist of just joining the cut ends, without any other consequence than what was initially desired, ie remove a defect -a mutation- of this gene or completely prevent its expression. But the cut is often imperfect and ends up by the removal or the omission of one DNA base that is sufficient to prevent the correct reading of the repaired DNA. Several bases may have been lost, others may be inserted in the opened DNA. An entire piece of cut DNA can sometimes be re-inserted in the cut-off site, in the right direction or reversed, or even in two copies. Two genes opened at the same time can be inadvertently "glued" together, sometimes even on two different chromosomes. These examples illustrate how the DNA repair machinery is naturally not perfect, which disturbs the normal reading of our genome, sometimes leading to the generation of abnormal or even malignant cellular elements. This happens naturally and a fortiori when using the CRISPR/Cas 9 system.

The promises of a perfect future system lie into precision improvement

Designing the best guides is the first key challenge that we can address to limit the off target risk. Since the guides provide the precise targeting of the gene of interest, the choice of the guides sequences is essential to ensure that they will only be attached to the area to be cut. Improving the accuracy of the CRISPR system implies that of guide selection tools, through very reliable bioinformatics methods.

In-depth analysis of edited cells genome is key

Since it is not possible to control cell repair mechanisms it is therefore mandatory to monitor off-target events. This can be done by in-depth analysis of the genome of edited cells, through various techniques including DNA sequencing. This allows to evaluate the number and exact location of any unwanted events before transferring modified cells into the patient, and thus stop a clinical protocol deemed too risky compared to the benefit it would bring.

Delivery methods allows to monitor the expression duration of Cas9 nuclease

Delivery methods of the CRISPR system into the cells, or the entire organism are also widely investigated. Controlling the nuclease can be performed via its transient expression: the enzyme will be present into the cells just enough time to perform the requested cut and then it will disappear. Non integrative cell transfer of the Cas9 nuclease is a very promising and safe approach. In this regard, FlashTherapeutics has developed the Lentiflash® technology, a new lentiviral vector dedicated to the delivery of RNA into any cell, combining the advantages of transient expression and the wide cell penetration of lentiviral vectors. Such technology makes it possible to monitor the expression of Cas 9 and to respond to a safety consideration essential for therapeutic use, even in the case of in vivo administration, as a vaccine.

CRISPR/Cas9 remains a great hope, all over the world, to cure many diseases

If the perfect tool does not exist yet, it's a safe bet that we get closer every day. The research around the CRISPR system goes extremely fast and arouses an enthusiasm never seen before since they constitute a great hope to cure many diseases like genetic disorders but also cancer or viral diseases like AIDS.

 

 

Christine Duthoit  Project leader Cell engineering & Immunology Flash Therapeutics expert since 2009