by hearingearly hearingearly

Exosomes are expected to break through the current barriers of CAR-T cell therapy

CAR-T was successfully prepared by exosomal delivery of mRNA

In 2012, Emily, a six-year-old American girl, experienced two relapses of acute lymphoid leukemia and was in danger of dying. In this case, Emily was transferred to the Children’s Hospital of Philadelphia, received CAR-T treatment, experienced all kinds of tribulations, and finally 23 days later, the doctor found that Emily’s cancer has completely disappeared, and became the first CAR T therapy in history to cure leukemia children.

In recent years, the world has set off a boom in the development of CAR-T therapy, “120 thousand needles”, “anti-cancer god drugs”, “two months of cancer cells zero”, but also let CAR-T therapy was once “deified”, has become a “lifesaving hair” in the eyes of many patients and their families.

The full name of CAR-T Therapy is Chimeric Antigen Receptor (CAR) T-cell Therapy, also known as chimeric antigen receptor T-cell therapy, which is the human T cells modified in vitro by genetic engineering means, and then transfused back into patients for the treatment of diseases. CAR-T therapy originated in 1989, when renowned scientists Gross G, Waks T, and Eshhar Z, in a study, chimed a gene sequence expressing a specific antibody into cytotoxic T cells (CTL), which gave T cells the ability to recognize the antigen – 2,4, 6-trinitrobenzene (TNP). This enables T cells to achieve antigen-specific, non-MHC-restricted activation and enhancement of their effects. “Giving T cells a chimeric antigen receptor will be an important way to fight cancer in humans in the future,” the three scientists said in their study.

At present  , the FDA has approved a total of six CAR-T therapies, and two CAR-T products have been listed in China, and a total of eight CAR-T therapies have been approved worldwide!

With the continuous development of CAR-T technology, clinical results show that CAR-T cells have great advantages in the treatment of hematological malignancies. However, with the deepening of research and increasing application, the adverse reactions caused by CAR-T therapy have gradually been recognized and paid attention to.

Currently, most CAR-T therapies are prepared by lentiviral transduction, but safety concerns may arise due to the immunogenicity of lentiviruses and the uncertainty of insertion sites. In addition, CAR T therapy with lentiviral transduction has many obstacles in production and in vivo transformation, such as high cost, limited capacity, and in vivo immunogenicity.

Compared with lentiviral transduction, the use of mRNA to induce CAR expression can allow CAR expression in a short period of time, avoiding the risk caused by prolonged existence in the body. Optimization of CAR mRNA structure can make CAR protein more easily translated and expressed in T cells, and its efficacy is no less than that of CAR-T therapy generated by lentiviral transduction. The current common technique for delivering mRNA to T cells is electroporation, but high-voltage electrical pulses can cause permanent damage to the cells, affecting the expansion and lethality of CAR T cells.

In February 2023, A paper published by Chinese researchers titled “Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric. antigen receptor T-cell engineering article on the use of exosomes to deliver mRNA for the preparation of CAR-T therapy.

Exosomes are secreted from living cells into the extracellular environment and contain a variety of biologically active molecules, which are absorbed by corresponding receptors and mediate intercellular communication. Exosomes are relatively stable in structure and can effectively protect mRNA from degradation. Previous studies have shown that exosomes derived from induced pluripotent stem cells can effectively protect mRNA from degradation even below 4℃. Exosomes have lower immunogenicity, better biocompatibility and can cross the blood-brain barrier. In addition, exosomes can be engineered to enhance their targeting.

The researchers used the phage coat protein MS2 to load specific mRNA into exosomes and demonstrated that the coated mRNA molecule was able to be translated into the protein in the recipient cell. N-terminal fusion with lysosomal associated membrane protein subtype 2B (LAMP-2B) enables the expression of CD3/CD28 single chain variable fragments (ScFv) on extracellular membranes. Finally, the MS2 binding site was inserted into the 3 ‘UTR of the CAR gene to obtain engineered exosomes that could be used directly for T cell activation and CAR T cell preparation.

In the experiment, the researchers confirmed that CD3/CD28 ScFv on exosomes was more easily bound to T cell membranes, and observed that CAR-T constructed using exosome delivery mRNA had good killing efficacy. This may be an alternative strategy to deliver mRNA to T cells more safely and efficiently, and also provide new ideas for the preparation of CAR-T in vivo.

In the article, the author also mentioned the challenges of using exosomes to deliver mRNA to prepare CAR-T therapy, such as the efficiency of loading mRNA into exosomes. It has been reported that the fusion of proteins that can bind specific RNA sequences to exosomes can improve the efficiency of mRNA entering exosomes. In addition, lipid-coated purified mRNA is produced by pre-incubation with cationic lipids, and then incubated with exosomes to produce exosom-liposome hybrids carrying mRNA, which can also effectively improve the efficiency of mRNA entering exosomes.

After nearly 30 years of development, CAR T cell therapy has become a major emerging pillar in the field of tumor treatment, and this transformation may be expected to turn “anti-cancer” into “cancer treatment”, ushering in a new era of fighting cancer.

by hearingearly hearingearly

Exosomes | The most complete interpretation

I. What are exosomes

Exosomes are small vesicles secreted by cells, with a diameter of 30-150 nm, which contain cell-specific proteins, liposomes, and complex RNA, which naturally exist in body fluids and can be transmitted to other cells as signal carriers to change the function of other cells, so as to regenerate cells. Exosomes were first identified in sheep reticulocytes in 1983, and were named “exosome” by Johnstone in 1987. In 2013, three scientists from the United States and Germany discovered the “regulatory mechanism of exosome transport” and won the 2013 Nobel Prize in Physiology or Medicine.

II. The formation and composition of exosomes

Many kinds of cells can secrete exosomes in normal and pathological state. The production process of exosomes is as follows: the membrane invaginates to form endosomes, then forms polyvesicles, and then the polyvesicles fuse with the cell membrane and are released outside the cell to form exosomes. The rate of exosome production in different cells is different, and the size and composition of exosomes are highly heterogeneous, and the composition is closely related to the source cell.

III. the role of exosomes

1. Intercellular information transmission of exosomes

Exosomes are widely present and distributed in various body fluids, just like postmen, carrying multiple “letters”, such as proteins, mRNA, miRNA and lipids, etc. As important signaling molecules, exosomes form a new cell-cell information transmission system, which can participate in cell communication, cell migration, angiogenesis and tumor cell regulation and other processes.

Specific to the application of treatment, such as: can effectively promote the healing of fractures, protect and repair kidney damage, myocardial weakness and hind limb ischemia treatment, as well as regulate and promote the regeneration of skin, muscle, hair follicles and other tissues and organs.

2. Immune system regulation of exosomes

The study of exosomes began with the study of immunology-related mechanisms, and it was found that exosomes are particularly closely related to immune regulation: exosomes are loaded with various immune-related molecules, through which the immune response is regulated.

Exosomes derived from immune cells contain antigenic peptides – MHC complexes and various antigens, which can significantly inhibit tumor growth, activate T cells and B cells, and may also promote the expansion of CD4+T cells.

Similar properties could be used to develop novel immunotherapies that could eventually lead to a cure for cancer.

Exosomes, which promote immunosuppression, can effectively inhibit inflammatory factors and have strong anti-inflammatory ability, which can be applied to the treatment of patients with inflammation or autoimmune diseases.

3. Exosomes are used as drug carriers

Exosome – extracellular vesicle drug delivery system has the advantages of high biocompatibility, low immunogenicity, intracellular targeting, and easy chemical and genetic manipulation. It can be used as an integrated platform for multiple drugs or therapeutic strategies to synergistically activate multiple steps of the tumor immune cycle, thus being widely used to amplify the anti-cancer immune response.

Exosomal drug delivery technology surpasses the technical barriers existing in traditional T cell-mediated cellular immunity (tumor antigen release, antigen presentation by antigen presenting cells (APC), T cell activation, recruitment and penetration of T cells in tumors, and recognition and killing of tumor cells by T cells).

4. Exosomes are used in disease diagnosis

The mechanism of exosome production suggests that by analyzing the composition of exosomes, it can help identify the cell type from which they originate. This property has been used to develop molecular diagnostic methods for cardiovascular diseases, central nervous system diseases, and tumors, and has also been valued in the diagnosis of liver, kidney, and lung-related diseases. Other studies have shown that tumor-derived DNA fragments carried in exosomes can help detect tumor-associated DNA mutations.

In addition, exosome-related mirnas have high value in tumor diagnosis, such as miR-100, miR-21, etc. In addition to mirnas, some exosomal proteins may also be used for molecular diagnostics.

In recent years, the clinical research of exosomes by Chinese researchers has shown explosive growth, showing great application potential in disease treatment, and the progress of modern biomedicine represented by exosomes has been unstoppable.