CRISPR Protocol: Inhibition of mTOR by means of CRISPR / Cas9 as an alternative to regulate the proliferation of cancer cells.


Proliferation is an important part of cancer development and progression. This is manifested by the expression and / or altered activity of proteins related to the cell cycle. The constitutive activation of many signal transduction pathways also stimulates cell growth. Once tumors appear, growth and metastasis can be supported by the overproduction of appropriate hormones (in hormone-dependent cancers), by promoting angiogenesis, by the transition from epithelium to mesenchymal, by triggering autophagy and by take the signals of the surrounding stromal cells. Cancer therapy, which involves cytotoxic drugs, destroys cells that have a high basal level of proliferation and regeneration. While this type of therapy targets tumor cells, it affects non-tumor cells that proliferate rapidly. The objective of this study is to program through CRISPR-Cas9 a sequence to genetically inhibit the proliferation pathway of the rapamycin Diana protein in mammalian cells (mTOR) to alter and block the proliferation of tumor cells by coupling it to the target cells by electroporation medium.

The mTOR protein is involved in the control of the initiation of RNA transcription, organization of the actin cell cytoskeleton, membrane traffic, ribosome formation and growth regulation, cell proliferation.

The two proteins that interact with mTOR, raptor and rictor, define the different arms of the mTOR path. The raptor-mTOR pathway regulates cell growth (accumulation of cell mass) through S6K1 and 4E-BP1. It responds to nutrients and growth factors, partly due to regulators such as TSC1 / TSC2 and rheb. The rictor-mTOR complex regulates Akt / PKB, PKCα, Rho / rac, to control cell survival, proliferation, metabolism and cytoskeleton. The binding of growth factors to receptors on the active PI3K cell surface, to generate PIP3, which recruits the PKD1 and Akt / PKB kinase to the membrane. Akt / PKB is activated by phosphorylation in two different places. The rictor-mTOR complex phosphorylates Akt / PKB in serine 473, in a hydrophobic motif, which facilitates phosphorylation by PKD1 in the Akt / PKB activation loop in threonine 308.

Neoplastic cells require a large cocktail of cytokines for cell cycle control, proliferation and metastasis. Inhibitors directed against the metabolic pathways generated by these cytokines are generating promising results in the treatment of solid tumors and leukemia. As mentioned earlier, the mTOR protein seems to be an essential protein that is in the PI3K signaling pathway. On the other hand, there is solid experimental evidence that reinforces the role of mTOR as a key regulator of the cell cycle, directing expansion, growth and proliferation.


Generate a vaccine using CRISPR Cas9 to couple an interference sequence to P42345 that represents the mTOR genetic code.


An interference sequence will be designed for the initiators and part of the mTOR coding sequence in order to couple it to the CRISPR-Cas9 system and generate a selective genetic inhibitor of cell proliferation.

To evaluate the effectiveness of the designed system,

Use of Oncolytic Viruses and Immunostimulation Therapy

For several years now, HSV-1 has been used in the biotechnology industry as a vector for transfection of eukaryotic cells and has also been used in different clinical trials. In 2007, researchers from London reported that they treated successfully (although modestly) a rare form of blindness caused by a mutation in a single gene (the gene encodes a protein that is important for producing pigments that absorb light in the eye). Most importantly, the patient did not suffer any side effects. More recently, researchers in France used a modified HSV-1 to introduce genes to correct a rare brain disorder (adrenoleukodystrophy) in two children.

Oncolytic viruses (VO) use cancer cells to replicate and selectively destroy altered tissue spreading within the tumor, without damaging normal tissue. In addition to this direct oncolytic activity, VOs are also very effective in inducing immune responses in infected tumor cells.

The VOs encompass a wide variety of DNA and RNA viruses that are selective for cancer or can be genetically modified. The VOs provide a diverse platform for immunotherapy; they act as vaccines in situ and can be armed with immune modulating transgenes or combined with other immunotherapies such as administration of Dendritic cells and Tumor Infiltrating Lymphocytes (TIL-NK)

Many of the characteristics of the neoplastic tissue provide a permissive environment for oncolytic viruses.

OVs have many features as an advantage in different therapeutic uses:

1.- Cancer cells cannot develop resistance to Oncolytic Viruses

2.- They replicate selectively in tumor cells are not pathogenic.

3.- There is a possibility that the dose of virus in the tumor increases over time.

4.- Safety features can be incorporated, such as drug sensitivity and immunity or Viral Neutralization mechanisms.

An important issue for therapy with Oncolytic Viruses is either systemic or local administration. With intravenous systemic administration multiple tumors can be attacked, however, intratumoral administration is more effective and the number of virions in the tumor tissue is increased in less time.

There have been numerous clinical trials of VO for cancer. As expected, most have been in phase I with some phase II trials. There is currently a phase III trial of an oncolytic HSV1 for melanoma (talimogene laherparepvec, T-Vec) of this Visur strain there is the final analysis showing results that will lead to the first approval by the US FDA. UU. From a VO for tumors.

Like many cancer vaccination strategies, our collaboration with the team of researchers from the National Institutes of Health has developed a type of OV that apart from generating lysis of cancer cells leaves an antigen in the survivors (for patenting ATC ) that can induce selective adaptive immune response in the body’s defense cells. The combination of the expression of ATC Known Tumor Antigens in the tumor and OV-mediated cell death induces enhanced migration and activation of T cells compared to infected tumor cells that only express viral antigens. This is coupled to a main vaccine strategy (which contains a VSH-1 blocking agent which limits the replication of the virus used.

The presence of HSV-1 has been detected worldwide. It is acquired mostly during childhood and lasts a lifetime. The vast majority of HSV-1 infections are cold sores (infections in the mouth or around them, so it is sometimes called cold sores, labial) without any vital commitment.

The viruses and their code were initially developed in plasmids to transfect bacterial cells and produce as many viruses as possible, subsequently tested with renal adenocarcinoma cell lines (769-p), squamous cell carcinoma (UPCI: SCC152) and corroborated that the virus replicated and generated lysis of cells cultured in vitro, we are currently carrying out tests with hepatocarcinoma and small cell lung carcinoma cell lines, to demonstrate their oncolytic activity.

Finally, we will evaluate the oncolytic activity in a murine model to be able to translate the technology to a phase 1 clinical study in humans, with this background our work group is in the task of innovating and developing new tools aimed at the well-being of our patients and the population with genetic predisposition to solid neoplasms.