Cell Report: The gene that awakens “silence” from “in vivo.”

Hearing disability ranks first among all types of disabilities in China, of which 60% of deafness is caused by genetic factors. Gene therapy is the golden key to the treatment of hereditary deafness caused by genetic mutations, especially the genetic repair of DNA, and it is given the hope of “curing the root cause”.

On July 12, The Cell Report published the latest progress by Xiong Wei’s team and collaborators, a researcher at the Beijing Center for Brain Science and Brain-Like Research (formerly associate professor at the School of Life Sciences at Tsinghua University), who for the first time demonstrated in vivo gene therapy for congenital genetic diseases using the gene repair pathway of non-homologous terminal junction (NHEJ) in mammalian models.

From treating the symptoms to the root causes

According to the World Health Organization’s website, approximately 466 million people worldwide suffer from disabling hearing loss, including about 34 million children.

Clinical treatment of hereditary deafness, mainly cochlear implants and hearing aids, as well as chemotherapy, but these can not fundamentally treat deafness.

Gene drugs are the foundation after chemical drugs and the key direction of the next generation of drugs developed by clinical institutions, and the prospect of gene therapy for genetic diseases is becoming more and more clear in the case of technical thresholds such as human gene delivery and gene expression.

Although gene therapy is seen as a “savior” for the treatment of genetic diseases caused by genetic mutations, most of the current research efforts focus on gene substitution or gene silencing for mRNA.

Xiong Wei said in an interview with China Science News that mRNA-based gene therapy, although a number of target genes with good applicability have been used in the clinic, reflecting the feasibility of gene therapy, but as far as the central law is concerned, repair at the mRNA level is still a symptom, and gene repair for DNA is a new direction for the next generation of gene therapy.

He explained that DNA is like the “source code” of life, mRNA, etc. are “products” of DNA transcription, and in terms of diseases caused by genetic mutations, gene substitution or gene silencing for mRNA is only the treatment of “products”. Among them, gene substitution is mainly to add to the mutant genes, the original mutant genes lead to loss of function, and replace them by foreign supplementation of wild-type genes; Gene silencing is done by subtraction to eliminate the disease-causing mutation gene.

In his view, gene therapy for mRNA is not regulated by the body’s endogenous sources, is instantaneous, and will “fail” after a certain period of action.

“Gene repair brought about by DNA-oriented gene editing can enable the repaired genes to follow the growth rhythm of living organisms and achieve endogenous expression regulation in time and space, which is the most natural embodiment of gene expression and regulation, and it is also the most ideal way to repair genetic diseases.” Xiong Wei said.

After nearly 7 years of research, Xiong Wei’s team finally proposed a protocol specifically for DNA code-shifting mutations, and systematically demonstrated that gene editing at the DNA level of cochlear hair cells can effectively restore animal hearing.

They also analyzed and evaluated the in vitro and in vitro animal levels from multiple perspectives such as editing gene products, protein expression, hair cell function, and animal physiological function to demonstrate the feasibility of the protocol.

From the outside of the body to the inside

In recent years, more and more researchers have devoted themselves to the exploration of gene editing technology, but the in vivo gene therapy program based on gene editing technology has been in the laboratory and cell line system research stage.

Xiong Wei told reporters that DNA-level gene repair needs to rely on gene editing technology represented by CRISPR-Cas, and with the development of CRISPR-Cas tools in 2011, researchers can rely on guide RNA (gRNA) to achieve stable and controllable fixed-point DNA double-strand cutting/breaking (DSB).

There are many pathways for DSB remediation, the most important of which are non-homologous end connection (NHEJ) and homologous recombinant repair (HDR). NHEJ products carry various insertion or deletion (InDel) resulting in codeshift mutations, and its results are equivalent to from one mutation to multiple mutations, so gene therapy is not well used.

The HDR-dependent repair pathway is to repair the target gene to the wild type, but this process requires a wild-type DNA template, but in vitro cases, the repair efficiency based on the HDR pathway is very inefficient on the terminal differentiated cells of living animals, which limits the application of gene editing in gene therapy to some extent.

Since 2016, a succession of cell line-based studies have shown that the terminal ligation products of DSBs produced by gRNA-spCas9 enzyme cleavage are not random, but can be predicted and repeatedly achieved.

Based on this principle, the DNA editing events obtained by a given gRNA can be predicted, and conversely, the gRNA can be selected according to the needs of the DNA product. But the actual situation of this law in the body is not yet known. Xiong Wei said.

In this study, they used a strain of Pcdh15av-3j mouse strains that mimic human DFNB23 genetic deafness to systematically demonstrate that DSBs generated near mutation sites can be repaired by the NHEJ pathway to repair codeshift mutations and partial repair of auditory and equilibrium functions.

This study involves verification at both the organizational and in vivo animal levels. Gene delivery of cochlear hair cells has always been a difficult point at the tissue level.

In 2014, Xiong Wei’s team developed a systematic solution for cochlear tissue culture and gene electrotransmission, on the basis of which they first scanned the editing products generated by 4 gRNAs in the 15 bp region upstream and downstream of the av-3j mutation (1 bp insertion), and found that the m-3j-gRNA1 showed a high proportion of 1 bp deletion editing products, and the reproducibility was high. This means that m-3j-gRNA1 is a potential candidate gRNA for the treatment of av-3j mutations.

On the cultured Pcdh15av-3j cochlear tissue, they also electrotransformed SpCas9 and m-3j-gRNA1 Pcdh15av-3j hair cells to restore the expression of PCDH15 protein, and the mechanical transduction function of Pcdh15av-3j hair cells was also restored.

The co-first author of the paper, Liu Lian, a graduate of Tsinghua University, told reporters that according to the effects of in vitro research, the team further carried out experiments at the somatic animal level, injecting AAV2/9 wrapped in m-3j-gRNA1 in pcdh15av-3j mice in the cochlea.

Through the systematic comparison of gene editing products of electrotocochlear tissue (in vitro) and viral injection of cochlear tissue (in vivo), it was found that they were very consistent in both the ratio of the main edit product and the product.

They further evaluated various indicators of m-3j-gRNA1 virus injection mice, including PCDH15 expression of cochlear hair cells, mechanical transduction current of cochlear hair cells, auditory brainstem potential and startle response in mice, all of which showed that a single m-3j-gRNA1 could achieve functional recovery of av3j mutation with the help of spCas9.

This use of mouse cochlear culture tissue to verify that the functional on-cell editing product of terminal differentiation is also reproducible, and uses this principle to achieve a single gRNA to repair the code-shift mutation and realize in vivo gene therapy in mice. The successful validation of the concept suggests that genetic diseases caused by codeshift mutations, which account for 22% of humans, have broad therapeutic prospects.

Advance scientific research “down-to-earth”

Since the establishment of the Neural Communication Laboratory at Tsinghua University in 2015, Xiong Wei has led a team to study the physiological and pathological mechanisms of deafness-related genes, and has also experienced the difficulty of “starting from scratch”.

In addition to all the technology and resources to accumulate from scratch, various strategies and methods were tried, and finally the non-random repair mechanism of NHEJ was used to reshape the deaf mutation caused by the transcoding mutation.


Xiong Wei (fourth from left) team Courtesy of the interviewee

This year, Xiong Wei joined the Beijing Brain Science and Brainoid Research Center. He admits that he has also imagined a failure to verify, the body is a complex environment, some experiments are very good in vitro, but in the body may be completely changed, “if the verification fails, it can only be said to be pushed down again.” ”

Xiong Wei told reporters that the current policy on the application of gene editing in the human body is still unclear, mainly the unknown nature of new technologies, how to use within the prescribed range, and the discussion of reducing possible harm to a minimum is very necessary.

He revealed that in addition to the code shift mutation, they are also conducting tissue and in vivo research to replace the mutation. “Next, if conditions permit, we will further conduct experiments in pigs and non-human primates, and advance scientific research on the ground under the conditions of policy.” Xiong Wei said. (Source: China Science Daily Tian Ruiying)

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