The gene editing revolution

With the rapid devolution of CRISPR/Cas-9 techniques, the modification of the genomic material has become very easy and accessible. The potential of gene editing include not only biopharmaceutical development, but also agro-research and the prevention of viral-transmitted diseases.

The CRISPR/Cas-9 technique of gene editing has been indicated by the scientific magazine Science as the “Breakthrough of the Year 2015” technology¹. The method allows for the modification of the genomic material of living organisms, with a very broad set of potential application. Just as an example, the first experiments on the human embryo were authorised at the beginning of 2016 at the Francis Crick Institute in the United Kingdom in order to better understand the genes involved in the correct foetal development.

CRISPR/Cas-9 techniques were first published in 2012: their rapid diffusion is connected with the very simple procedures and flexibility of application and to the low costs compared to other gene modification techniques: no more than 30$ are sufficient to run a basic experiment, according to Nature².

The many advantages and the ethical concerns of gene editing have been deeply discussed by the scientists attending the International Summit on Human Gene Editing in December 2015: the final Statement highlights the risks also associated to the use of CRISPR/Cas-9 techniques on the human being and the need to urgently reach a wide social consensus towards their application to manipulate the human embryo for clinical applications. Jennifer Doudna, one of the inventors of the new method, reported on Nature³ her nightmares about the potential impact CRISPR/Cas-9 might have if improperly used.

Human embryo’s manipulation

The first scientific paper describing the genetic modification of non-vital human embryos was published on Protein & Cell⁴ in April 2015 by a group of the Chinese Sun Yat-sen University at Guangzhou: gene editing was used in triploid zygotes cells to correct the gene responsible for sickle-cell anemia. «The Chinese researchers used the embryos which were unuseful for in vitro fertilisation procedures as they had an abnormal DNA content, incompatible with the normal development», tells Paolo Vezzoni, who is managing the Institute of Genetic and Biomedical Research (IGBR) of CNR at the Humanitas Hospital in Milan. The CRISPR/Cas-9 technology has no dark sides, the expert further tells Pharma World. The issues under debate refer to its application only on somatic cells or on germline cells too. If the genetic modification, for example, is made just on the patient’s blood cells, which are then re-implanted into its body, the patient is the only person who need to face the safety issues. «If a germinal cell line is used, the DNA of the modified cell is transmitted to the entire organism, included the newly formed oocytes and the spermatozoa. The same correction will be transmitted to the descendants of the patient», explains Vezzoni. Thus, if a damage occurred with the modified DNA, it will be inherited by the new generations. Before CRISPR/Cas-9, the modification of the genomic content of the human embryo was too difficult to make this theoretical possibility to become real.

How it works

The application of the “clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease” (CRISPR/Cas-9) represents a revolution for molecular biology. Cas-9 genes are coding for enzymes (helicases and nucleases) responsible for the site-specific cut of DNA sequences. The natural repairing machinery is then recruited to repair the damage.

CRISPR/Cas-9 techniques allow to exactly cut the DNA at the point where the new gene should be inserted; the old, damaged genetic material may be removed, or it is inactivated if left in place. «The estimates tell that with a 5-10% correction of the genetic defect, the genetic disease improves and becomes less severe», tells Paolo Vezzoni.

The method could turn very useful also for the delivery of gene therapies, an issue not yet fully solved and which may deeply affect the availability of new treatments for rare diseases. According to Paolo Vezzoni, there are three main obstacles to the full availability of gene therapies: the first bottleneck is represented by the method used to deliver the gene inside the cell. The possible choice falls between viral or not-viral vectors. «Then it is necessary to control the gene once it entered the cell. Finally, it is difficult to target the gene only to the damaged cells. It is not easy, for example, to insert a gene into the cells of the central nervous system. CRISPR/Cas-9 techniques have greatly helped in solving these issues», tells Vezzoni.

A further problem is the possible occurrence of a casual insertion of the new gene, causing an improper reading of the genomic information by the translation machinery, which may result in a further deterioration of the function of the organism. «Leukemia or tumours might develop in such instances», highlight the expert. The technique might be useful, for example, to deactivate the genes responsible for the rejection of pig’s organs in xeno-transplants.

The impact of gene editing technique may also involve the modification of the genome of animals and plants, something which may alter the natural balance between different species in the ecosystem. The method is under study to prevent the transmission of infective diseases, such as malaria, the Dengue fever or ZIka, by mosquitoes. Another field of interest for the application of gene editing techniques is the creation of transgenic or knock-out animals for pharmaceutical research, or of modified plants able to grow under particular environmental conditions or to resist to insects and infesting plants.

The industrial potential

According to the data published by Nature², the Massachussetts Institute of Technology is currently the leader as for the number of patents on CRISPR/Cas-9 technologies (62), followed by the Broad Institute (57) and the MIT’s bioengineer Feng Zhang (34). Among the industry, the agriculture-focused multinationals Danisco (29) and Dow Agrosciences (28) are deeply investing in the field. Many pharmaceutical and biopharmaceutical companies have already closed deals and research agreements to use gene editing platforms for the discovery and development of new therapies.

The use of CRISPR/Cas-9 techniques in agriculture may proceed slower than its pharmaceutical application because of the debate on the status, genetically modified or not, of the plants obtained with gene editing. «The method allows for the modification of the plant’s genome without insertion of new genes – explains Vezzoni. – It could be argued that the plant should then not be considered a genetically modified organism. The possibility to eliminate noxious insects is very interesting from the theoretical point of view, but it is impossible to exactly evaluate its impact once applied in the real world». The entire ecosystem might be affected by the introduction of modified mosquitoes or flies, and it is yet very difficult to exactly define the sustainability of such kind of intervention⁵, which for example might be very useful for the prevention of malaria. Many in vivo experiments with genetically modified mosquitoes are on going, and we will further discuss them in a following issue of Pharma World.

The International Summit Statement on human gene editing

The recent world congress on gene editing ended with the issuing of a final statement resuming the conclusions of the Organizing Committee on the ethical and societal concerns emerged from the debate.

1. Basic and preclinical research. Research should proceed, subject to appropriate legal and ethical rules. Critical issues have been identified on the technologies used for editing genetic sequences in human cells, the potential benefits and risks of proposed clinical uses, and the understanding the biology of human embryos and germline cells. The modified cells should not be used to establish a pregnancy.
2. Somatic clinical use. Many promising clinical applications are directed at altering genetic sequences only in somatic cells. There is a need to understand the risks, such as inaccurate editing, and the potential benefits of each proposed modification. Proposed clinical uses can be appropriately and rigorously evaluated within existing and evolving regulatory frameworks for gene therapy.
3. Germline clinical use. Important issues include the risks of inaccurate editing and incomplete editing of the cells of early-stage embryos (mosaicism), the difficulty of predicting harmful effects that genetic changes may have, the obligation to consider implications for both the individual and the future generations. Furthermore, once introduced into the human population, genetic alterations would be difficult to remove and would not remain within any single community or country. Permanent genetic ‘enhancements’ to subsets of the population could exacerbate social inequities or be used coercively. It would be irresponsible to proceed with any clinical use of germline editing unless and until the relevant safety and efficacy issues have been resolved, and there is broad societal consensus about the appropriateness of the proposed application.
4. Need for an ongoing forum. While each nation ultimately has the authority to regulate activities under its jurisdiction, the human genome is shared among all nations. The international community should strive to establish norms concerning acceptable uses of human germline editing and to harmonize regulations, in order to discourage unacceptable activities while advancing human health and welfare. The forum should be inclusive among nations and engage a wide range of perspectives and expertise.

Source: The National Academies of Sciences, Engineering and Medicine

Who invented gene editing?

Three different scientist are claiming the invention of gene editing techniques: Jennifer Doudna from the University of California Berkeley, Emmanuelle Charpentier, the current director and scientific member at the Max Planck Institute of Infection Biology in Berlin, and McGovern’s and Broad Institute’s researcher Feng Zhang.

The first article describing the method⁶ was signed in 2012 by both Doudna and Charpentier. The scientist from Berkeley’s University has also been the first to claim intellectual protection. Other observers⁷ identify Feng Zhang as the first inventor of CRISPR/Cas-9 techniques. A priority dispute is pending at the U.S. Patent and Trademark Office among Doudna and Zhang: something which could turn very important from the point of view of the commercial exploitation of the technology.

The first biotech company focused on gene editing was the spin-off of the Berkeley’s University Caribou Biosciences, founded by Jennifer Doudna and Rodolphe Barrangou. The company has closed collaboration agreements with DuPont for agricultural applications and with Novartis for the screening and validation of new drug targeting systems.

Intellia Therapeutics was founded in 2014 to develop cellular and genetic therapies; the company is collaborating with Novartis. The new division eXtellia Therapeutics was established in 2015 for the development of ex vivo therapeutics.

Doudna and Zhang together founded in 2013 Editas Medicine. The company has signed an exclusive collaboration agreement with Juno Therapeutics.

CRISPR Therapeutics was founded in 2014 by Emmanuelle Charpentier and Daniel Anderson and has established collaboration agreements with Cellgene Corp., Bayer, Généthon and Vertex Pharmaceuticals.

References

1. Science 18 Dec 2015: Vol. 350, Issue 6267, pp. 1446-1448, doi: 10.1126/science.350.6267.1446
2. Nature 522, 20–24 (04 June 2015) doi:10.1038/522020a
3. Nature 528, 469–471 (24 December 2015) doi:10.1038/528469a
4. Protein & Cell 6, 363–372; 2015
5. Science 08 Aug 2014: Vol. 345, Issue 6197, pp. 626-628, doi: 10.1126/science.1254287
6. Science 17 Aug 2012: Vol. 337, Issue 6096, pp. 816-821, doi: 10.1126/science.1225829
7. Cell, Vol. 164, Issue 1-2, pp.18-28, 14 January 2016, doi: 1016/j.cell.2015.12.041