Technological Overview

Introduction

The Company develops products allowing fine and precise genome engineering of any cell or biological organism.

The technology of the Company, protected by a large portfolio of patents and patent applications, relies on exploiting the natural mechanisms used by all living organisms for repairing their DNA. For maintaining the integrity of their genome, all cells use a DNA maintenance and repair system (the repairosome). The technology of the Company allows the activation of this repairosome in a precise and targeted manner, at the same time, resulting in the reprogramming of the DNA in a corrective manner.

This technology opens the route of the rational engineering of cells and other living organisms, as well as that of genome surgery for human therapy.

The Company designs protein "scissors" capable of cleaving the Deoxyribonucleic Acid (DNA) at precise sites in living cells. It commercialises "systems of recombination by meganuclease" (MRS: Meganuclease Recombination Systems). An MRS is a product made of "scissors" and a DNA matrix allowing to target a defective gene in any living organism (human, plant, micro-organism) or cell and to substitute by another gene in a precise and controlled manner. The DNA sequence variations amongst species (for example, in the human genome and in that of maize) make an MRS operate only in the species and on the gene for which is was designed.

The MRSs of the Company are the first real "cut-and-paste" of the DNA in living cells, following the example of the famous word-processing tool.

History of the Technology

In that context, the Company has obtained licences from the Institut Pasteur on patents and patent applications protecting mainly two fundamental technologies: homologous recombination and use of intron endonucleases. These technologies qualify as being fundamental as they describe and explain phenomena seen in nature. The first technology (homologous recombination) consists of replacing one gene with another one by using identical sequences upstream and downstream of the gene. The second (use of intron endonucleases) consists of using the natural properties of molecules which cut DNA at an exact location.

These two technologies in themselves allow the realization of only a limited number of operations mainly within the scope of research and for certain industrial applications.

Moreover, the Company has perfected and protected a technology (Meganuclease Recombination System or "MRS") allowing the cutting of DNA in a predefined region thanks to modified endonucleases called "meganucleases". This represents a breakthrough in comparison to technologies licenced by the Institut Pasteur, which are based upon a finite list of endonucleases which, by definition, only allow cutting at a single region in a given DNA. The Company's technology thus permits the development of artificial endonucleases capable of cutting DNA at any desired location defined in advance.

Several of the Company's clients already use MRSs based upon natural intron endonucleases. Notably, the products are already being used for the deletion of undesirable sequences in plants^[1] and the engineering of strains for industrial production^[2].

[Figure A] : Principle of the cleavage carried out by a meganuclease and of the DNA repair by means of a targeted DNA matrix with respect to reprogramming the genome of the cell.

Products Applications

The construction of specifically modified meganucleases to target a gene or a sequence chosen a priori presupposes that the sequence is known. The genomic revolution of the 90s is characterised by the sequencing of the human genome and has been followed since by genomic sequence data from several living organisms and microorganisms. The number of known genomic sequences is growing rapidly and, with it, the number of potential specifically modified meganuclease targets (tens of millions). Specifically modified meganucleases have a large range of potential targets.

In the bioproduction field

In the biotechnology field of medication manufacture, reprogramming is performed on a small number of cells that, after the procedure, will divide rapidly. Each cell gives birth to two other cells carrying the same genome as the original cell. Thus it is only necessary to modify the original cells within a population in order that the correction or insertion successfully spreads throughout that population.

In the field of therapeutic protein production, the genetic engineering which the meganucleases enable constitutes an important advance on several fronts.

1. Simplifying the production process and reducing turnaround time

   The random approach used until now limits the efficacy of cellular engineering as a means of producing proteins. The use of meganucleases targeting insertions at always the same location in the genome will allow homogenous expression clones to be obtained in a reproducible manner which are stable long-term.

2. Improving the quality of therapeutic proteins

   The quality of therapeutic proteins is determined by the cell line. Meganucleases make it possible to alter the expressing cell line's metabolism to obtain therapeutic proteins with modified functions (by acting primarily on post-translational mechanisms).

By way of example, an expressing cell line could be modified to prolong the half-life of a therapeutic protein which would enable an increase in its efficacy and reduce the risks of immune reactions linked to repeated injection of the product.

The industrial stakes of these applications are high and there are numerous known molecular targets.

In the agronomics field

In the vegetable kingdom, few classical approaches can meet the needs for improving agronomical traits and, in particular, the need for a rapid, rational and non-random intervention. The main seed growers (Monsanto, Dupont, Dow, BASF, Bayer, Syngenta and Limagrain) are actively working on targeting methods in plants. The inactivation of genomic sequences, the targeting of regions chosen a priori, the substitution of different versions of the same gene all represent examples of applications to which specifically modified meganucleases are particularly suited. Besides, the number of species contemplated (corn, rice, soya, rape, alfalfa, etc.) is very large even if they do not all have the same economic potential.

By way of example, certain genes in corn can be knocked out in order to limit residual biomass in the production of bioethanol.

In the biotherapeutics field

In the biotherapeutics field, hundreds of potential biotherapeutic targets exist which play a clear role in certain clinical conditions and which can not be targeted or controlled by pharmaceutical compounds. Similarly, numerous diseases result from a gene deficiency and only its restoration can treat the condition.

Alternative therapies are therefore required to correct genomic targets which are refractory to classical pharmalogical methods. This situation could enhance the clinical prognosis of patients afflicted by these conditions but also commercial opportunities for therapeutic meganucleases. Meganucleases used for performing genomic surgery would allow the substitution of "deficient" sequences carrying inherent deleterious mutations (monogenetic diseases such as immunodeficiencies like X-SCID, skin cancers linked to Xeroderma pigmentosum, etc.) or undesirable viral sequences with "healthy" sequences. This approach consisting of targeting the genetic cause of the condition and treating it by means of genomic surgery constitutes a completely novel medical intervention.

In the research field

Finally, specifically modified meganucleases are likely to find many applications for use in research, notably in the study of gene function. Although this area offers more limited commercial opportunities, it is the basis for establishing the Company's innovative technology as a worldwide standard in DNA modification.

Cellectis' Products

[Figure B] : Structure and function of meganucleases. I-CreI and I-SceI are naturally occurring intron endonucleases. DemoCre is a synthetic hybrid meganuclease.

A meganuclease is produced according to its DNA sequence. That sequence is inserted into the genome of a living organism (bacterium), which assimilates it as information for producing the molecule. The Company has at its disposal a very small-scale production capacity which enables it to supply small quantites of molecules to its clients and partners. If the client or partner wishes larger quantities, it shall need to put in a request to the Company, which then supplies the DNA sequence encoding the molecule. The client or partner can then, either approach a subcontractor who has a large-scale production capacity, or use their own production capacity. All these procedures are protected by a patent and patent applications filed by the Company.

Thus the Company either delivers small quantities of molecules in a test tube, or the encoding sequence of that molecule as a data file to its clients and partners.

The repair matrix is a DNA sequence which encodes the gene that the client or partner wishes to insert in place of the targeted gene. The Company either delivers a small quantity of DNA produced under its care, or the theoretical sequence that will then be produced by the client or by a service provider.