Genome Engineering

The principle of genome engineering is simple: it involves modifying the genetic heritage of an individual or species for the purposes of understanding, production or treatment. It’s about applying genetic research conducted at the end of the last century. Genetics has demonstrated the link between the physical attributes of species and their genes. Genome engineering enables species’ genes to be modified in order to modify their attributes, to rectify an error or to add a new advantageous trait. This approach is not a new one: cross-breeding different plants or selecting the best animals for reproduction is based on this same principle. The aim is to create individuals with the best possible attributes – corn with more grains per ear or cows that produce more milk.

The targeted approach of genome engineering is predictable and more reliable and effective than directed reproduction. The genomes of over 1,000 organisms have now been decoded. Our knowledge of the sequence and location of the genes of living beings is therefore constantly improving; however, the workings of these genes continue to hold secrets for us. Our understanding in this field allows us to manipulate genes directly. There are three possible modes of action:

• Insertion: We know the gene responsible for the attribute that we want to add, and we want to insert it into a genome. For example, GMOs – plants are given the capacity of producing a specific insecticide.
• Inactivation: We want to prevent the expression of a gene in an individual. For example, in fundamental research, we want to know the role of a given gene in an organism.
• Correction: We want to replace an existing erroneous sequence (which generally causes the gene to be defective) by a functional sequence. For example, in medicine, to remedy a genetic disease.

Cellectis uses very powerful tools known as meganucleases to carry out these operations. There are also other techniques which prove the potential of genome engineering applications. The sector is thriving because it has applications in a wide range of areas.

Meganucleases

The effective power of genome engineering compared with previous technologies lies in its ability to insert the gene of interest in the right place. For a long time, researchers’ methods were akin to playing darts blindfolded; they would study the effect of randomly inserted sequences, despite the fact that insertion in the wrong place can have harmful effects – one gene can be inserted into another and inactivate it, or activate cancer mechanisms. This is where Cellectis has a competitive edge. Its expertise in the area of meganucleases means that it is able to precisely target, and therefore manipulate, a particular region of the genome. Cellectis provides scientists with the precision they were lacking.

Meganucleases are natural proteins found in many single-celled organisms. They are highly specific “DNA scissors”. They are able to recognize their binding site by identifying a nucleic acid series (nucleic acids are the constituents of DNA) which contains from 12 to over 30 base pairs and which is statistically unique in a genome. Once the break has been made, the cell activates its maintenance and repair system (homologous recombination). This system corrects the DNA molecule by using its twin copy as a model, for example.

Cellectis bases its work on these properties to develop its various genome engineering applications using meganucleases. The process involves introducing a meganuclease that is specific to the targeted site into the cell and inserting a gene with the attributes required to stimulate homologous recombination. The meganuclease breaks the DNA molecule and the homologous recombination system corrects this break by taking as a model the gene introduced at the same time as the meganuclease. It is the same principle as “copy and paste” in word processing.

Protein engineering

The key for Cellectis is to engineer meganucleases that are specific to the targeted site, so as to create “tailor-made” meganucleases for each gene of interest. This ability to produce a wide range of molecules specific to the genes targeted is the technological gamble which led to the creation of Cellectis. The gamble paid off in 2004, when the company’s researchers achieved the industrial-scale modification of the recognition site of an existing meganuclease, without the resulting new molecule losing its ability to break the DNA and stimulate recombination.

Within the structures of the known meganucleases, the researchers identified the areas responsible for the “DNA scissors” function and the areas that correspond to the recognition of specific DNA sites. Cellectis then modified this latter part so as to generate variants that retain their cutting activity but bind to a different region of the genome. Cellectis currently has a large bank of proteins with over 20,000 motifs that can be combined to obtain chimeric proteins capable of binding to various regions of the DNA molecule.

When a new project requires a new meganuclease so that a different site in the genome can be manipulated, Cellectis’ teams create a meganuclease from this protein bank. In the same way as a child’s building blocks, a red brick and a yellow brick together create a new construction. The bricks are the parts of the genome that code for the various target areas. Scientists identify the molecules whose targets are close to the target aimed for. They then take the corresponding areas on the genome and combine them to create a new molecule with the specific recognition site required. The activity of each newly created meganuclease is tested in a series of functional trials developed by Cellectis.

Research & Development

One of our company’s foremost assets is the strong position of its intellectual property estate. The Institut Pasteur patent families covering the use of homologous recombination and the use of endonucleases from the meganuclease family to induce DNA recombination formed the initial basis of Cellectis’ founding. We have also since developed a healthy proprietary portfolio. Cellectis patents fall into three broad categories – patents on general principles, patents on specific meganucleases and applications, and patents covering the manufacturing of modified meganucleases. At the end of 2009, Cellectis owned the rights to 53 patent families, including 58 patents granted and over 200 patent applications. In 2009 alone, Cellectis was granted four new patents and filed 15 new patent applications. Our strategy involves building up an intellectual property portfolio that protects our products, their applications and the corresponding body of technological knowledge.

The company strives to optimize and uphold the intellectual property it licensed from Institut Pasteur. Cellectis also endeavors to promote its own intellectual property by defending its own rights, while respecting the rights of others.