JP-2022512792-A5 -

Dates

Publication Date

20221223

Application Date

20191023

Description

This invention relates to an animal model of dwarfism with an IGF-1 gene mutation and a method for producing the same. This invention was developed with support from the Korean government under project number KGM4251824, "Development of General-Purpose/Custom-Made Artificial Blood Using Miniature Pig Resources," of the Ministry of Science, ICT and Future Planning, Korea. The main characteristic of Laron syndrome is abnormally short stature (dwarfism), and physical symptoms include a complex phenotype such as a prominent forehead, small skull, mandibular developmental disorder, trunk obesity, sparse hair, bone developmental abnormalities (teeth, skull, thigh), saddle nose, double chin, small genitals, achondroplasia (small hands and feet), and delayed puberty. The cause of these symptoms is mainly related to mutations in the growth hormone (GH) and GH receptor genes, and consequently, a decrease or deficiency of IGF-1 is known to lead to these symptoms. Therefore, in the development of treatments for Laron syndrome, it is necessary to develop animal models that reflect a phenotype similar to that of Laron syndrome so that the efficacy and safety of the treatments can be evaluated. Until now, rodents have been almost exclusively used as disease models for drug treatment and mechanistic research of genetic disorders. However, the pathological features and symptoms of animal disease models differ significantly from those observed in humans, leading to numerous problems when conducting clinical trials based on results from rodent disease models. In other words, currently, no animal model has been established that expresses all the symptoms of human genetic diseases to the extent that it can be used as an effective disease model. Therefore, due to the significant differences in anatomy/physiology, reproduction, lifespan, and behavioral patterns between humans and animals, there is a growing need for disease animal models using species that are closer to humans. This has led to increased demand for utilizing pigs, which can be used in research on intractable diseases, as a new animal model in the field of biopharmaceuticals. To date, the IGF-1 knockout mouse is a model animal developed for Laron's disease. However, it has been reported that IGF-1 knockout mice die within one day of birth due to respiratory distress. To overcome this, a mouse model has been developed in which IGF-1 is specifically knocked out in the liver, which is responsible for 75% of IGF-1 production, using a Cre/loxp conditioned system. While this mouse model exhibits a 75% or greater reduction in circulating IGF-1 levels in the blood, it has the problem of exhibiting no phenotypic difference from normal mice. Recently, transgenic cloned miniature pigs were produced by knocking out the growth hormone receptor gene using genetic scissors. However, only some of the phenotypes seen in patients with Larons' disease (dwarfism, obesity) were observed. In other words, a model animal that expresses all the symptoms of Larons' disease to the extent that it can be used as an effective disease model has not yet been established. Therefore, the inventors conducted research to develop transgenic animals exhibiting diverse phenotypes that appear in dwarfisms such as Laron syndrome, and completed the present invention. KR10-2009-0051716AKR10-2018-0091291AWO01-72119A2 This figure shows the IGF-1 nucleotide sequence into which the CRISPR/Cas9 gene scissor guide RNA (gRNA), located in exon 2 of porcine IGF-1 , hybridizes .This figure shows the structure of the porcine IGF-1 reporter vector in the IGF-1 knockout gene scissors Surrogate Reporter System.This figure shows the structure of the porcine IGF-1 gRNA vector in the IGF-1 knockout gene scissors Surrogate Reporter System.This figure shows the structure of the Cas9 vector in the IGF-1 knockout gene Scissors Surrogate Reporter System.These are fluorescence micrographs verifying the introduction and operation of the IGF-1 knockout gene scissors expression vector system.This graph shows the results of flow cytology analysis to verify the introduction and operation of the IGF-1 knockout gene scissors expression vector system.This figure shows the IGF-1 nucleotide sequence mutations (#3 to #21) to which gRNA hybridizes in normal miniature pig donor cell lines (WT) into which the IGF-1 knockout gene scissor expression vector system has been introduced.This figure shows the changes in amino acid codons of the IGF-1 gene due to sequence insertion in IGF-1 knockout (KO) transformed donor cell #7. The parts shown in red are stop codons.This figure shows the changes in amino acid codons of the IGF-1 gene due to sequence insertion in IGF-1 knockout donor cell #15. The parts shown in red are termination codons.This figure shows the results of karyotype analysis of IGF-1 knockout (KO) transformed donor cell #7.This figure shows the results of karyotype analysis of IGF-1 knockout (KO) transformed donor cell #15.This is a s