动物实验 z-bo 2020-09-05 450

　　The Tmc1 gene is necessary to detect the normal function of the hair cells of the inner ear sound waves. Humans and mice with specific dominant mutations in the Tmc1 gene experience progressive hearing loss. In a new study, researchers used the CRISPR-Cas9 genome editing strategy to inactivate the mutant form of the gene, thereby reducing deafness in a Beethoven mouse model. "This is a very important study," said Peter Bargill Spey, a sensory biologist at Oregon Health Sciences University (he did not participate in this study). This Beethoven mouse model has the same point mutation in the mouse version of the human Tmc1 gene. After about 3 weeks, these mice began to lose their hearing. By eight weeks, due to the death of hair cells, he was deaf and dumb.

　　In this new study, Harvard chemical biologist David Liu and his colleagues are the autosomal dominant pathogen of the gene. Instead of using a virus-based system to transfer Cas9 and gRNA sequences, we designed a guide RNA (gRNA) that specifically targets gender copies, but a ribonucleotide protein (RNP) called Cas9-gRNA complex. . Encapsulated in lipids and transferred, the strategy is to increase the mutant allele by 20 times compared with the wild-type allele in mouse fibroblast cultures to increase the mutant allele Editing optional.

　　Next, these researchers injected the lipid-encapsulated RNP complex into the inner ear of a newborn mouse model of Beethoven (including mutant and wild-type alleles) and injected it. Only the unreceived inner ears were used as internal controls. These injected ears had intact and healthy hair cells, while the uninjected ears had rapid hair cell death by 8 weeks. Liu said: "This is exciting because it shows that the injected ears can remain healthy and enriched in hair cells compared to the uninjected ears." These researchers used the auditory brain to monitor the sexual response (ABR) test 4 Hearing in a Zhou-old Beethoven mouse model. ABR is used to measure the response of neurons to sound. The ABR recorded by the uninjected ear is about 75-80 decibels. This is equivalent to the noise level of waste disposal. The injected ear can hear a quiet sound of about 60 dB. This corresponds to a quiet conversation. This is an improvement, but wild mice can hear sounds as low as 30-40 dB. This shows that CRISPR genome editing can partially avoid hearing loss. By 8 weeks, the ABR threshold of the injected ears was still lower than the non-injected ears, but above the 4-week ABR threshold, these mice continued to be deaf. It is recommended that the behavioral responses to loud sounds follow a similar pattern between these treated and wild mice.

　　Barr-Gillespie said: “Although this is far below the ABR threshold of wild mice, the 10-15 dB gap will have a significant impact on humans., Neuroscientist Brigitte Margrange, University of Liege, Belgium (not involved in this study) John · Ulrich Müller, a young neuroscientist at Hopkins University School of Medicine, said (the treatment of hereditary hearing loss is an impressive and very important result) (not involved in this research). So the next step will be manual testing." He explained that many hereditary deafness occur during the development of human fetuses, which makes this delivery strategy of genome editing more difficult. However, despite some common problems, the RNP complex delivery strategy developed by these researchers still has the potential for off-target effects compared with delivery strategies that rely on persistent viral infection. cut back. This is one of its advantages. "As long as the genome editing reagents can be effectively implemented, they can be delivered immediately."

　　Liu said. If this is a complex delivery of a local RNP, there are some practical benefits. "