CRISPR against cancer: details
CRISPR/Cas9 components for cancer treatment were delivered to the tumor using lipid nanoparticles
Ekaterina Gracheva, "Elements"
Fig.1. Genetic editing using the CRISPR/Cas9 system. The Cas9 endonuclease (depicted as two light blue ovals) finds a section of the genome in which it is required to make a directed genetic modification using an artificially synthesized guide RNA (guide RNA). It includes a section of crRNA, complementary to the target sequence, as well as trans-activating RNA (tracrRNA), which are interconnected by a linker (broken line). The DNA sequence PAM (protospacer adjacent motif) is also involved in the recognition of the target sequence. Cas9 endonuclease cuts DNA along both strands. The resulting double DNA break can lead to deletion in the target DNA region and destruction of the target gene. Illustration from the website mirusbio.com .
The CRISPR/Cas9 genome point editing system is gradually being adapted for use in medicine. Including for the fight against cancer. A recent study has shown that CRISPR/Cas9 can be used to target cancer cells. To do this, the components of the system configured to cut out the PLK1 gene were placed in special lipid nanoparticles that were injected into tumors induced in mice. Such therapy has shown good results against both glioblastoma and ovarian adenocarcinoma, in both cases significantly increasing the lifespan of mice and reducing the size of tumors.
The use of "molecular scissors" of the CRISPR/Cas9 system, which allow point-to-point editing of the genome, gave a new impetus to research in genetic engineering. This year, pioneering work on the study of this system and its implementation in practice was awarded the Nobel Prize. Elements has repeatedly talked about this system and its various applications. For example, it has been used to obtain thousands of cell lines and laboratory animals with specified properties.
In addition, genomic editing with CRISPR/Cas9 has a huge therapeutic potential, including for fighting cancer. The CRISPR/Cas9 system is already being tried to be used for the treatment of certain diseases. While these works are at the stage of clinical research, but it is quite possible to hope that their progress will not take long. So, in the spring of 2020, it was announced (H. Ledford, 2020. CRISPR treatment inserted directly into the body for the first time) about the first introduction of the CRISPR/Cas9 system directly into the retina of a patient suffering from Leber's amaurosis, a hereditary disease that leads to vision loss. And more recently, the New England Journal of Medicine published the results of successful correction of mutations in patients with beta-thalassemia and sickle cell anemia (H. Frangoul et al., 2020. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia).
The use of CRISPR/Cas9 for the treatment of oncological diseases is based on the same idea as that of chemotherapy: to reduce the activity of proto–oncogenes - genes that usually regulate cell division, but for some reason (due to a mutation that increases the activity of a protein product, gene duplication or for other reasons) becomes overactive. One of the important proto–oncogenes is the PLK1 gene (polo-like kinase 1). The product of this gene is a kinase that activates proteins necessary for mitosis. Overexpression of this gene is observed in glioma, melanoma, breast, prostate, ovarian carcinoma cells and many others (Z. Liu et al., 2017. PLK1, A Potential Target for Cancer Therapy). Clinical trials of the drug Volasertib (Volasertib), which blocks the work of PLK1 kinase in patients with various malignant tumors, are currently underway.
Chemotherapy has a number of disadvantages. Firstly, several rounds of drug administration are required, and since they do not act selectively (that is, not only on cancer cells), healthy cells also suffer. Because of this, the patient repeatedly experiences the entire spectrum of adverse events (vomiting, hair loss, anemia, etc.). Secondly, some cancer cells become resistant to chemotherapy over time. This is due to the fact that chemotherapeutic drugs trigger an evolutionary process in the tumor: there is a selection of cells that are able to survive in their presence. Adapted tumor cells can inactivate the drug, prevent it from entering the cell, or simply not respond to it, disabling the control systems for the state of the cell (for example, the response to DNA damage). A similar process can be observed in the occurrence of resistance of microorganisms to antibiotics. Therefore, getting rid of the overexpression of an important gene in a cancer cell once and for all is a great idea, and the CRISPR/Cas9 system can just help in this.
The authors of the study, led by Professor Dan Peer from Tel Aviv University, used a very simple approach: they tried using the CRISPR/Cas9 system to destroy the PLK1 gene that promotes excessive proliferation of tumor cells. The system they designed consisted of two RNA molecules (this is one of the standard implementations of genomic editing based on CRISPR/Cas8 systems). The first molecule is a matrix RNA that encodes the Cas9 nuclease. The second molecule is a guide RNA (single guide RNA, sgRNA), which will lead the nuclease to the PLK1 gene, after which it will make a double–stranded break in the right place, which will lead to the destruction of the gene.
Then it remains to solve only one problem: to deliver these RNAs to the tumor cells. One of the possible ways is to use lipid nanoparticles (Fig. 2). They consist of positively and neutrally charged lipid molecules that form a complex with negatively charged nucleic acids. Such a "bubble" with a parcel of RNA or DNA interacts with the negatively charged membrane of living cells and merges with it. After that, nucleic acid will be released into the cytoplasm, which, depending on the purpose, does its job. This method has proven itself well in cases where it is required to deliver small molecules of nucleic acids to cells. Here, scientists needed to deliver quite large RNA complexes.
Fig. 2. Preparation of lipid nanoparticles (lipid nanoparticle) containing the CRISPR/Cas9 system. To assemble the nanoparticles, an alcohol solution of various lipid molecules (lipid mixture in ethanol), a matrix RNA encoding the Cas9 nuclease (Cas9 mRNA), and a guide RNA (sgRNA) corresponding to the target gene were used. The assembly was carried out in the NanoAssemblr microfluidic system. An image from the article under discussion in Science Advances.
The authors began by searching for lipid molecules for nanoparticles-carriers. They selected several suitable molecules from the library developed by the same scientific group (see S. Ramishetti et al., 2020. A combinatorial library of lipid nanoparticles for RNA delivery to leukocytes) and tested first their ability to form a complex with RNA, and then the ability of the complex to deliver Cas9/sgPLK1 RNA to cells. Of the four options considered, which differed from each other in the linker part, the one that formed stronger bonds with RNA molecules was chosen.
The authors also modified the Cas9 matrix RNA with 5-methoxyuridine to make it more stable. Also, this modification helps to avoid the immune response of the body to the introduction of foreign RNA.
For the work, the authors selected two types of tumor cells corresponding to the most dangerous types of oncological diseases. Cells of the first type, the so–called 005 line, were obtained from mouse glioma artificially induced by the introduction of activated oncogenes h–RAS and Akt (T. Marumoto et al., 2009. Development of a novel mouse glioma model using lentiviral vectors). When these cells are introduced into the body of mice, aggressive glioblastomas are formed. The life expectancy of a person with such a diagnosis is only 15 months, and the five–year survival rate is only 3%.
The second cell line, Ovcar8, was obtained from a high–grade ovarian adenocarcinoma. These cells are resistant to a large number of chemotherapy drugs, and when injected into the body of mice, they form numerous metastases. Ovarian cancer is often detected only at the stage of metastasis, when the chances of survival are significantly reduced. Therefore, the search for effective therapy against it is a very important task.
Cultures of 005 and Ovcar8 cell lines were successfully modified using a newly developed system: the PLK1 gene was destroyed in 84% of 005 cells and in 91% of Ovcar8 cells. 48 hours after the addition of nanoparticles, cell division was suspended, and after 96 hours, cell viability significantly decreased.
This result was very encouraging, but before testing the nanoparticles on real tumors, it was necessary to check how well the mice tolerate their introduction. Healthy mice were injected with lipid nanoparticles of the same composition, but they contained RNA complexes aimed at destroying the green fluorescent protein (GFP) gene. Mice do not have this gene, so such particles can be used as a control. A day after administration, a general blood test of mice was performed, and the level of liver enzymes was measured (to check the toxicity of nanoparticles) and the level of pro-inflammatory cytokines reflecting the degree of immune response. No significant deviations from the norm were observed in mice. But the authors emphasize that in the case of preclinical studies, this aspect should be studied in more detail.
Finally, nanoparticles with "anti-cancer" RNAs were injected into mice.
First, the system was tested on induced glioblastoma. Mice were injected with a suspension of 005 cells into the hippocampus, and after 10 days, either a system directed against the PLK1 gene or against GFP (control) was injected directly into the resulting tumor. Two days after the introduction of Cas9/sgPLK1 RNA, the PLK1 gene was damaged in ~68% of cells. And three days after the injection of nanoparticles into the tumor, a significant number of its cells showed signs of apoptosis. At the same time, the non-dividing neurons surrounding the tumor, where the activity of the PLK1 gene was already low, remained alive.
Finally, a group of 30 mice with induced glioblastoma were injected with Cas9/sgPLK1 RNA nanoparticles (either control in the form of a standard sodium-phosphate buffer solution or nanoparticles with Cas9/sgGFP RNA) and their survival was studied, as well as tumor size reduction was measured. 30% of mice that received an active RNA complex were alive 60 days after tumor induction. All animals in the control groups died by the 40th day. The average survival rate increased from 32.5 to more than 48 days, and the tumor size decreased significantly.
After such success, the authors moved on to work with ovarian adenocarcinoma cells. This was necessary not only to check whether the system works on other tumors, but also to test its capabilities. For example, is it possible to direct action against tumor cells.
For the treatment of some tumors, such as metastatic or malignant diseases of the hematopoietic system, the drug has to be administered systemically so that it is distributed to all target cells. The obvious disadvantage of this method, which has already been mentioned above, is that chemotherapy affects the entire body. Delivery systems based on lipid carriers have another problem: a lot of lipid nanoparticles end up in the liver due to the peculiarities of metabolism (A. Akinc et al., 2010. Targeted Delivery of RNAi Therapeutics With Endogenous and Exogenous Ligand-Based Mechanisms). That is why genetic modification of liver cells using CRISPR/Cas9 is usually quite successful, reaching ~70% (J. Finn et al., 2018. A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing). To modify cells in other organs, either injection directly into the tumor is required, or some kind of trick so that genome editing occurs only in the target cells.
Earlier, the authors of the study developed a method that can solve this problem (N. Viega et al., 2018. Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes). In the lipid particles carrying nucleic acids, they included another component – lipoprotein, which is able to interact with an antibody. This antibody, in turn, interacts with a protein characteristic of a particular type of cell (Fig. 3).
Fig. 3. The scheme of assembly of lipid particles with a motif for targeted delivery. The lipid nanoparticles include micelles with artificial lipoprotein (yellow-red). The protein part of this lipoprotein is able to interact with antibodies to the receptor of the target for the introduction of lipid particles of cells (red triangle). Illustration from the article N. Viega et al., 2018. Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes.
On the surface of ovarian adenocarcinoma cells, including Ovcar8 cells, there is an EGFR protein – an epidermal growth factor receptor. It is also present in other cells of the body, but there is especially a lot of it on cancer cells. It is this protein that antibodies are found, which are attached to lipid nanoparticles with Cas9/sgPLK1 RNA. Mice were injected with a suspension of Ovcar8 cells into the abdominal cavity to form tumors, and after 10 days, lipid nanoparticles with an antibody to the EFGR receptor were also injected there. Two days after administration, the PLK1 gene was damaged in ~82% of cells. Modified cells, as well as glioblastoma cells, died by apoptosis, and the overall survival of mice with a tumor increased by about 80%.
These results allow us to say with cautious optimism that a new system has been obtained to combat oncological diseases. Firstly, it can be configured to modify various oncogenes using a properly similar guide RNA. Moreover, this way it is possible not only to destroy genes, but also to correct mutations. Secondly, it is possible to use antibodies that will be suitable for other types of tumors. For example, some types of breast cancer are characterized by overexpression of the HER2 receptor, which can be used as a target for a guiding antibody. Thirdly, the authors showed that for a good result, one injection of lipid nanoparticles with Cas9/sgPLK1 RNA is sufficient. This is an important observation for the development of therapy for brain tumors. The blood–brain barrier is an obstacle to the delivery of chemotherapy to such tumors. Of course, it is possible to inject therapy directly into the tumor, but repeated administration can be dangerous for patients. Therefore, a single injection of powerful therapy may be a good strategy for their treatment.
Finally, this system based on lipid nanoparticles can be used not only for the treatment of oncological diseases, but also for gene therapy in other cases.
It is worth noting that this is not the first study with an attempt to tame the CRISPR/Cas9 system for cancer therapy. In 2019, a similar study was published, where the system was used to destroy the lipocaine-2 gene, which is associated with the growth of breast cancer cells (see P. Guo et al., 2019. Therapeutic genome editing of triple-negative breast tumors using a noncationic and deformable nanolipogel). However, the authors used a different version of the delivery system, and instead of RNA, they used plasmids encoding Cas9 and directing RNA against the lipocaine-2 gene. The effectiveness of the modification was 53%.
Source: Rosenblum et al., CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy // Science Advances. 2020.
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