CRISPR Mediated Knock out of Immune Checkpoint Inhibitors for Cancer Therapy

CRISPR mediated knock out of Immune Checkpoint Inhibitors for Cancer Therapy

Cancer is a group of diseases characterized by abnormal cell growth, and is the first leading cause of human deaths in underdeveloped countries and the second in the developed world. According to 2015 statistics from the World Health Organization, approximately 9 million people die from cancer every year, and millions of new cancer cases are being diagnosed annually, revealing the frightening dimensions of the situation. Cancer treatment costs billions of dollars each year, aiming to improve the quality of cancer patient’s life. The current available treatments are not enough to cure the most types of this disease and effective in only a fraction of the cancer cases. Recently, development of targeted cancer immunotherapy has emerged as a new front in the battle against cancer that proved effective in extending the lifespans of cancer patients, and indeed cancer immunotherapy treatment has become one of the most important therapeutic technologies in the cancer treatment.

In cancer immunotherapy, patients' own immune system is directed to destroy tumor cells, thus ensuring the elimination of cancerous cells in human body. In this regard, antigenic factors found in cancer cells or other tumor- associated factors are being used to specifically target and destroy cancer cells by pointing immune cells towards cancer cells through antibodies against these molecules. Indeed, recent clinical trials have proven the effectiveness of PD-1 and CTLA-4 monoclonal antibodies in cancer immunotherapy. PD-1 and CTLA-4, found on surfaces of immune cells, conventionally function in immune check-points, thus preventing an autoimmune reaction to body’s own cells. However, tumor cells express the ligands for these receptors en masse, triggering a “friendly cell” response and escaping T-lymphocyte recognition and subsequent destruction. Antibodies against PD-1 and CTLA-4 bind to the corresponding receptors on the surface of T-lymphocytes, preventing T-lymphocytes from being inactivated and programmed to die by cancerous cells. Hence, cancerous cells can be more effectively destroyed by tumor infiltrating T-lymphocytes. Recent clinical trials that showed the effectiveness of PD-1 and CTLA-4 antibodies in cancer immunotherapy have changed the outlook of therapeutic pathways in cancer treatment, opening new horizon for future.

However, the current limiting factor is the cost of cancer immunotherapy, which is around $150,000 annually. Considering the fact that 350,000 new cancer cases are being diagnosed every year in Turkey, the high cost prevents the widespread adoption of this effective treatment regime. Additionally, since these antibodies are not presently produced in Turkey, making Turkey relying on international suppliers. The prohibitive cost and the closed-source nature of this treatment inspire scientists not just in Turkey but in many underdeveloped countries to look for the alternative solutions.

Scientists have been aiming for a long time to develop an efficient and reliable technology, so-called gene editing, to make targeted and precise changes in genomes. Though several gene editing technologies includingzinc-finger nucleases [ZFNs] and transcription-activator like effector nucleases [TALENs] have emerged they have not widely been adopted due to design challenges and high cost (2-3). However, in 2012, Doudna, Charpentier and their colleagues published a seminal paper, where they demonstrated that CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenescan be effectively used in gene editing (1). Compared to other available gene editing technologies the CRISPR/Cas9 approach proved to be cheap, fast and easy to design and use, enabling widespread use by many scientists from distinct fields.

In essence, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are used by certain bacteria and archaea to prevent invasion of bacteria by foreign genetic materials. Simply, Cas9 endonuclease forms a complex with guide RNA consisting of a crRNA (Cas-associated RNA) and a trans-activating crRNA (trRNA) which recruit the Cas9 effector complex to the target sequence, making double-stranded breaks (DSBs) in the genome in a sequence-specific manner. Specifically, a 20-nucleotide sequence in the genome complementary to crRNA together with an adjacent protospacer-associated motif (PAM) specific to the origin of the Cas protein acts as a GPS for Cas9 endonucleases.

CRISPR genome editing technology has recently come under the spotlight with blocking immunoregulatory control inhibitors PD-1 and CTLA-1 in mind. In summary, as a cheap and relatively more accessible alternative to antibody-based cancer therapies, CRISPR is tailored to knockout PD-1 and/or CTLA-4 receptor genes in T-lymphocytes. Clinical trials with CRISPR technology is about to start in the US and China, which are in competition to develop new technology. They aim to employ CRISPR to knockout of one or both PD-1 and CTLA-4 in the T cells and their preliminary results seems promising, suggesting that the strategy may work in our hands as well.

For this reason, five Turkish doctors and scientists, who obtained their PhDs from US, Ireland and New Zealand, have joined forces to knock out either PD-1 or CTLA-1 and together in T cells, aiming to reduce the cost of $ 150,000 per patient in cancer immunotherapy using CRISPR technology. In addition to genetically manipulating the genes in a patient’s own tumor infiltrating T-lymphocytes, we have also aimed to use the same engineered tumor specific T cells in different cancer patients by overcoming allogeneic effect, which is clearance of the transferred cells recognized as ‘non-self’ by the recipients. To achieve that, we have plan to perturb expression of HLA class I gene in the same cells using CRISPR approach, producing universal cancer specific T cells (4). After determining high frequent HLA alleles in Turkey population (5), we plan to produce highly efficient and durable cancer specific universal T cells by knocking out PD-1, CTLA-4 and HLA class I genes.

Dr. Oktay Kaplan, Lecturer&Researcher at Faculty of Medicine, Istanbul MedeniyetUniversity.

Osman Doluca, PhD,

KaanYılancıoglu, PhD

Cihan Aydın, PhD

CihanTastan

References

1-Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E.., (2012) Science, 337, 816–821

2-Santiago, Y. et al. Targeted gene knockout inmammalian cells using engineered zinc fingernucleases. (2008).Proc. Natl Acad. Sci. USA 105, 5809–5814

3-Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ, A TALE nuclease architecture for efficient genome editing (2011) Nat Biotechnol.; 29(2):143-8.

4-Torikai H, Reik A, Soldner F, et al. Towardeliminating HLA class I expressiontogenerateuniversalcellsfromallogeneicdonors.Blood. 2013;122(8):1341-1349.

5-Arnaiz-Villena, A., Karin, M., Bendikuze, N., Gomez-Casado, E., Moscoso, J., Silvera, C., Oguz, F.S., Sarper Diler, A., De Pacho, A., Allende, L., Guillen, J. andMartinez Laso, J. (2001), HLA allelesandhaplotypes in theTurkishpopulation: relatednesstoKurds, ArmeniansandotherMediterraneans. TissueAntigens, 57: 308–317.