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The United States will conduct CRISPR human clinical research to turn the "Pandora's box" into a "hanging pot for the world."

via:科学网     time:2019/9/4 22:31:39     readed:135

This isn't the first time gene-editing technology has gone into formal human clinical trials, but since EDIT-101 needs to be injected directly under the patient's retina, it is expected to become the world's first CRISPR/Cas9 gene-editing therapy used directly in humans.

In recent years, the use of CRISPR / Cas9 gene editing technology in the treatment of human diseases is in the ascendant. Today, how do we understand the significance of this technology to human beings? Is it a hanging pot for the world or Pandora's magic box?

Find a new way of "genetic scissors"

Leber congenital amaurosis (LCA) is a serious hereditary retinopathy and one of the main causes of congenital blindness in children. Infants with this disease lose binocular cone cell function rapidly from birth to under one year of age until they are completely blind.

Leeber's congenital amaustrus can be divided into many different types, most of which are caused by different gene mutations on autosomal chromosomes. As a result, researchers began to explore gene therapy for the disease for a long time.

At the end of 2017, Luxturna, the first gene therapy drug for LeBron's congenital black type 2, was approved for sale by FDA in the United States. Leeber's congenital black haze type 2 is caused by RPE65 gene mutation. The basic therapeutic principle of Luxturna is to deliver normal RPE65 gene to retinal cells through AAV virus vector. In March 2018, MIT Otolaryngology Hospital first gave human patients commercial therapeutic Luxturna injection. The patient paid $850000 for treatment at the time.

However, another type of Leber's congenital amaurosis, type 10, is unable to replicate this therapeutic approach. Because the cause of the disease is another mutated gene, CEP290. The coding sequence of CEP290 gene is larger than that of AAV virus. Simply put, a small transport vehicle can't deliver large normal genes to retinal cells - it's "overloaded".

The Eitas Medical decided to find another way, and they still hired the AAV virus, but it was not a normal CEP290 gene, but it could destroy the mutant gene's "gene scissors" _ S. aureus Cas9 (a CRISPR/ Cas9 gene editing tool). Under the guidance of the CEP290 specific guide RNA, the gene scissors directly delete or reverse the mutation sequence in the mutant intron in the light-sensing cells of the eye, thereby restoring the normal expression of the CEP290.

Prior to human clinical trials, the technology had undergone in vitro retinal tissue experiments, mouse experiments and primate safety experiments. The related papers were published in Nature Medicine in January 2019.

For the general public, the concepts of gene therapy and gene editing therapy may not be so easy to distinguish. "most of the gene therapy that has been used is achieved by introducing normal genes into the cell, which does not alter the original genome of the patient, and gene editing therapy, as a special category of gene therapy, uses CRISPR/ Cas9 gene editing tools to 'repair' the patient's own genes directly." Xue Tianxiang, a professor at China University of Science and Technology, explained in China Journal of Science. It can be seen that the treatment of Leeber's congenital black haze type 2 belongs to the traditional gene therapy, while the EDIT-101 therapy for Leeber's congenital black haze type 10 is the real gene editing therapy.

A skillful forerunner

In recent years, the study of gene therapy for ophthalmic diseases has been developed. According to incomplete statistics, the genes that have been approved by the relevant national health department for clinical treatment or are in clinical study include: the RPE65 gene, the CEP290 gene associated with the congenital melanosomes, the RPGR gene associated with the retinitis pigmentosa, the PDE6B gene, and the USH2A gene; The REP-1 gene associated with the absence of choroidal disease (a disease that has gradually lost vision from night vision and peripheral vision, resulting in a loss of vision and blindness), and so on.

"Ophthalmic diseases are a hot area of gene therapy." Qiu Zilong, a researcher at the Institute of Neuroscience, Chinese Academy of Sciences, told the Chinese Journal of Sciences, "Part of the reason is that the eye is a relatively independent organ in the human body. Because of the lack of large blood vessels, drugs injected under the retina are not easy to spread through the blood circulation to other parts of the body. Relatively speaking, the safety is more guaranteed.

In recent years, Xue Tian, Qiu Zilong and Zhang Mei, associate professor of China University of Science and Technology, have cooperated to explore gene editing therapy for retinitis pigmentosa by using a new gene editing technique called Targeted-RecA Enhanced homology-Directed repair (TRED). After gene editing, the degeneration of cone and rod cells in mice with retinitis pigmentosa was alleviated, and the photoreceptor function of retina recovered to a certain extent.

Unlike the EDIT-101, the TRED therapy not only destroys the mutant gene with the gene editing tool, but also promotes the homologous recombination of the mutation site by introducing the MS2-RecA complex protein system, thereby realizing the purpose of gene correction and cell function restoration.

"CRISPR / Cas9, just like its nickname'Gene Magic Scissor', is a scissors that can cut genes." "Comparing two gene editing therapies for ophthalmic diseases, EDIT-101 repairs abnormal gene splicing sites caused by gene mutations through gene editing, while TRED first cuts out abnormal genes and then repairs normal genes like patches," Qiu said.

In the current state of technology, the success rate of using the CRISPR/ Cas9 shear failure gene sequence is relatively high, and the success rate of the self-homologous recombination of the body is very low. In the view of Xue Tian, the treatment scheme of the EDIT-101 is aimed at the special genetic disease of the Leber's congenital black-type 10-type, which only needs to destroy a certain genome sequence, and skillfully avoids the common technical problem of low efficiency of homologous recombination and repair. Therefore, the EDIT-101 is well-suited as a guide for opening the gate of the CRISPR/ Cas9 gene editing therapy.

However, hereditary diseases such as Leber's congenital black spot type 10, which can be cured only by shearing abnormal genes, are few after all. "The vast majority of genetic diseases still need to work with CRISPR gene scissors and other repair tools. Therefore, how to improve the efficiency of repair is a problem that researchers must overcome in the future. Qiu Zilong said.

It's not a "Pandora's magic box" if it's in the right way.

EDIT-101 is not the first gene editing therapy approved for clinical trials.

As early as May 2018, Europe approved for the first time a clinical trial of gene editing in the treatment of β-thalassemia, the first human disease in Europe to be treated with CRISPR/ Cas9 gene editing. The treatment, called CTX-001, is to genetically edit the patient's hematopoietic stem cells in vitro and then transfer them into the patient.

Wu Yuxuan, a researcher at East China Normal University who participated in the study, told the Chinese Journal of Science: "Including a variety of eye diseases, beta-thalassemia, sickle-cell anemia and so on, the current clinical trials of gene editing mostly aim at the current incurable genetic diseases. If successful, they can overcome them." In order to benefit patients and society immediately, it can also feedback and promote the further development of gene editing research.

However, gene editing therapy seems to touch the sensitive nerves of the public more easily than traditional medical techniques. Some people regard gene editing as Pandora's Box and are uneasy that CRISPR/ Cas9, a genetic scissors, is moving from the laboratory to a wider range of human life.

In this regard, several experts interviewed said that it is necessary to strictly distinguish between gene editing of human embryos, germ cells and adult cells. The former will not only pass on heredity to the next generation and enter the gene pool of all human beings, but also affect all cells in individuals. This is not only unnecessary for the simple treatment of diseases, but also greatly increases the risk of anticipated adverse phenotypes. Therefore, in a visible period of history, it should be prohibited to carry out gene editing research on human embryos and germ cells.

"for now, gene editing therapy based on adult cells is a strategy worth advocating." "but only if the experimental design is strict, ethical issues are well considered, and the technical system is very perfect and mature," Wu said.

As we all know, there is a "miss" phenomenon in the process of CRISPR / Cas9 gene editing, that is, "scissors" cut in the wrong place. In theory, technological progress can only reduce the miss probability, but can not completely avoid the occurrence of miss.

However, Wu Yuxuan believes that as long as the design is rigorous and the previous verification experiment is complete, miss targeting will not be a great threat-the current gene editing strategies for clinical use are based on Cas9 RNP (the complex formed by the incubation of Cas9 protein and synthetic sgRNA in vitro). The Cas9 protein will degrade quickly after editing, and the probability of miss target will be greatly reduced. And even if miss occurs, in most cases there will be no side effects.

"All medical means are a trade-off between benefits and risks." "The important thing is that when a therapy goes to clinical trials and is directly used in humans, a professional ethical committee must be established to assess whether its potential risks are acceptable and acceptable," Xue said.

Related paper information:

Https://doi.org/10.1038/s41591-018-0327-9

https://www.nature.com/articles/s41591-019-0401-y

Https://doi.org/10.1126/sciadv.aav3335

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