The use of genetically modified animals for human diseases contributes to the understanding of the pathogenesis of various diseases and aids in the search for effective treatments. Consequently, there is an emerging trend of establishing appropriate genetic animal models capable of recapitulating the crucial phenotypes of human diseases to facilitate investigations about the pathogenesis and effective treatments. Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) technology demonstrated its successful application in genetically modifying the genomes of diverse species. Furthermore, CRISPR/ Cas technology has been established as a tool for producing genetic animal models that more faithfully mimic human diseases. In this review, we have focused on the application of CRISPR/Cas technology in developing innovative genetic animal models for the study of human diseases, specifically for the investigation of disease pathogenesis and treatment. We have also discussed the current technical challenges of CRISPR technology, such as off-target effects, and reviewed the emerging strategies developed to enhance its precision. Finally, we have provided an overview of the considerable potential of advanced tools derived from the CRISPR/dead Cas9 (dCas9) system, such as base and prime editors, and explore their future applications in creating more sophisticated disease models.

Fabry disease is an X-linked lysosomal storage disorder caused by GLA mutations, leading to a deficiency in α-Galactosidase A activity and subsequent accumulation of globotriaosylceramide (Gb3). This accumulation contributes to progressive multiorgan dysfunction, with cardiovascular complications, particularly endothelial dysfunction and left ventricular hypertrophy being major drivers of disease morbidity and mortality. Although enzyme replacement therapy is currently the standard treatment, its effectiveness is limited in addressing advanced cardiovascular pathology. To better understand Fabry-associated vascular and cardiac phenotypes, an isogenic human induced pluripotent stem cell (hiPSC) model in which GLA was knocked out was developed using CRISPR/ Cas9. GLA-knockout (GLA-KO) hiPSCs were differentiated into endothelial cells (ECs) and cardiomyocytes (CMs) to evaluate disease-relevant phenotypes in vitro . GLA-KO ECs exhibited normal morphology and differentiation capacity but showed markedly impaired tube formation, high expression of inflammatory genes ICAM1, VCAM1, and SELE, and increased mitochondrial and cytoplasmic reactive oxygen species levels. GLA-KO CMs demonstrated enlarged cell size and nuclear translocation of NFATC4, consistent with hypertrophic remodeling. Together, these findings recapitulate key features of Fabry vasculopathy and cardiomyopathy in a genetically defined, human-derived system. This platform enables direct investigation of Gb3-induced oxidative and inflammatory mechanisms and provides a valuable model for the preclinical evaluation of therapeutic strategies targeting the cardiovascular manifestations of Fabry disease.

The early-onset familial Alzheimer's disease (EOFAD/ FAD), the less common type of Alzheimer's disease (AD) currently affects a vast number of individuals worldwide. This type is being inherited as an autosomal dominant fashion. Missense mutations on Amyloid precursor protein (APP) and Presenilins 1 and 2 (PSEN1 & PSEN2) are known as major genetic factors in FAD. Conversely, missense mutations on microtubule-associated protein tau (MAPT) are also thought to involve. Up to date, several triple-transgenic animal models with muted forms of the human APP, PSENs and MAPT have been reported. Compared to other animals, canines are more emotional and their disease signs can be easily diagnosed. This attempt was to develop a triple transgenic canine model for the AD. We have obtained the coding sequences of APP, PSEN1 and MAPT from Dana-Farber/Harvard Cancer Center DNA resource core at HMS and incorporated several common AD mutations. The transgenic construct is composed of hNSE (ENO2) promoter-driven three AD genes fused together with modified 2A sequences. It was transfected into the canine fetal fibroblasts which were then used to perform somatic cell nuclear transfer (SCNT). The viable transgenic embryos were obtained after in vitro culture and the GFP was detected. In this study, we have successfully produced viable triple transgenic canine cloned embryos using SCNT technique. These transgenic canine embryos will be further developed into canines with FAD. The transgenic canines will be a good candidate in the AD research field.

해리슨 내과학에 의사에게 요구되는 세 가지 덕목 중 하나로 인간 이해를 소개하면서, 진정한 의사에게는 ‘Shakespearean breadth‘가 필요하다고 강조하였다. 최근 췌장 및 담도 질환 에서는 담낭 담석의 지속적 증가, 환자의 사연과 췌장-담도 질환과의 연관성, 자가면역 질환의 증가, 치료 후 높은 재발률 등 인간 이해가 요구되는 보고가 늘어났다. 이에 췌장 및 담도 질환의 발병기전을 인간의 습관 고리 및 적응 반응까지도 포함한 전체적이고 근원적으로 파악하여, ‘인간 이해를 고려한 췌장 및 담도 질환의 발병 모델’을 제시한다.

In the last several decades, cell therapy research has increased worldwide. Many studies have been conducted on cell therapy, and have revealed that transplanted cells did not survive for long, and implanted cells remained inactive causing immune rejection depending on the patient’s condition. Therefore, studies on cell-free therapy need to be conducted. To overcome these limitations, an alternative is the use of supernatant from cells, called “conditioned media (CM).” During in vitro cell culture, culture media supply nutrients to maintain cell characteristics and viability. In the culture, cells not only consume nutrients but also release beneficial proteins and substances, which are called “secretome.” CM from cells can be stored for a long time and is easy to handle. Moreover, secretome in CM can also be measured; exact amount of secretome is important to set the standard value for disease treatment. Here, we reviewed studies on CM and confirmed that various secretomes from CM were identified in these studies. Moreover, these findings could benefit cell and animal studies in future. In conclusion, CM could be a potential candidate for an alternative to cell therapy.

Deoxynivalenol (DON) and related trichothecene mycotoxins are extensively distributed in the cereal-based food and feed stuffs worldwide. Recent climate changes and global grain trade increased chance of exposure to more DON and related toxic metabolites in poorly managed production systems. Monitoring the biological and environmental exposures to the toxins are crucial in protecting human and animals from toxicities of the hazardous contaminants in food or feeds. Exposure biomarkers including urine DON itself are prone to shift to less harmful metabolites by intestinal microbiota and liver metabolic enzymes. De-epoxyfication of DON by gut microbes such as Eubacterium strain BBSH 797 and Eubacterium sp. DSM 11798 leads to more fecal secretion of DOM-1. By contrast, most of plant-derived DON-glucoside is also easily catabolized to free DON by gut microbes, which produces more burden to body. Phase 2 hepatic metabolism also contributes to the glucuronidation of DON, which can be useful urine biomarkers. However, chemical modification could be very typical depending on the anthropologic or genetic background, luminal bacteria, and hepatic metabolic enzyme susceptibility to the toxins in the diet. After toxin exposure, effect biomarkers are also important in estimating the linkage and mechanisms of foodborne diseases in human and animal population. Most prominent adverse effects are demonstrated in the DON-induced immunological and behavioral disorders. For instance, acutely elevated interleukin-8 from insulted gut exposed to dietaty DON is a dominant clinical biomarker in human and animals. Moreover, subchronic exposure to the toxins is associated with high levels of serum IgA, a biological mediator of IgA nephritis. In particular, anorexia monitoring using mouse models are recently developed to monitor the biological activities of DON-induced feed refusal. It is also mechanistically linked to alteration of serotoin and peptide YY, which are promising biomarkers of neurological disorders by the toxins. As animalalternative biomonitoring, huamn enterocyte-based assay has been developed and more realistic gut mimetic models would be useful in monitoring the effect biomarkers in resposne to toxic contaminants in the future investigations.

Smoking is a risk factor for oral leukoplakia and oral cancer, as well as lung cancer, cardiovascular diseases and many other systemic diseases. Smoking is considered increasing factor of some oral diseases involved indigenous bacteria. In addition, a relationship between smoking and infection of Human papillomavirus (HPV), which is associated with oropharyngeal cancer, remains unclear. The aim of this study is to assess whether smoking has an impact on increase of bacteria inducing oral disease such as dental caries and periodontitis, and HPV infection. DNA of saliva gathered from smokers and non-smokers, consisted of men and women, was analyzed using PCR. Oral disease-causing bacteria were more detected in men smokers than men non-smokers and HPV was most found in women non-smokers. Taken together, this study suggests smoking is related with variation of oral microorganism existence in some way.

Mesenchymal stem cells constitute an potential cellular source to promote brain regeneration with Parkinson's disease. Mesenchymal stem cells have significant advantages over other stem cell types and greater potential for immediate clinical application. The purpose of this study was to investigate whether hMSCs from the human adipose tissue could be induced to differentiate into dopaminergic cells and to assess the developmental potential of hMSC for selectively replacing the midbrain dopamine neurons lost in Parkinson's disease in vitro and in vivo. MSCs were cultured under conditions that promote differentiation of dopaminergic neuron. Using media that include SHH, FGF8, and GDNF. the MSCs were induced in vitro to become dopaminergic neurons. The expressions of the LIM homeobox transcription factor 1, alpha (Lmx1a), tyrosine hydroxylase(TH) proteins were determined by immunofluorescence. Lmx1a has been shown sufficient to confer neurogenic activity on mesencephalic floor plate cells and to determine a mesencephalic dopaminergic neurons fate. This result suggests that hMSCs have the ability to differfentiate into dopaminergic neurons. hMSCs were then transplanted into the striatal in a rat model of Parkinson's disease. The rats were unilaterally lesioned in the substantia nigra with 6-hydroxydopamine and were tested for rotational apomorphine-induced behavior. Following differentiation of dopaminergic neuron, cells displayed dopaminergic morphology and that they expressed dopaminergic marks genes. Finally transplantation of hMSCs into the striatal of Parkinsonian rats resulted in improvement of their behavioral deficits by apomorphine-induced rotational behavior. The hMSCs transplanted rats were proved to be better than compared with the transplantation of PBS. Immunohistochemical analysis of grafted brains revealed that abundant hMSCs survived from the grafts and some of them displayed dopaminergic marks. Our results indicate that hMSC may serve as a good cell source for the treatment of neurodegenerative diseases and have high potential for being used in multiple applications. This cellular approach might become a restorative therapy in Parkinson's disease.

The purpose of this study is to evaluate an efficacy of in vitro differentiated human embryonic stem (hES, MB03) cells expressing Nurr1 in relief of symptomatic motor behavior of Parkinson's disease (PD) animal models MB03 was genetically modified to express Nurr1 protein and was induced to differentiate according to 2-/4+ protocol using retinoic acid and ascorbic acid. The differentiation-induced cells were selected for 10 to 20 days thereafter in N2 medium. Upon selection, cells expressing GFAP, TH, or NF200 were 38.8%, 11%, and 20.5%, respectively. in order to examine therapeutic effects of the differentiated cells in PD animal model, rats were unilaterally lesioned by administration of 6-kydroxydopamine HCI (6-OHDA) into medial forebrain region (MFB, AP -4.4 mm, ML 1.2 mm, DV 78 mm with incision bar set at -2.4 mm), as a reference to bregma and the surface of the skull. Confirmation of successful lesion by apomorphine-induced rotational behavior, differentiated cells were transplanted into the striatum (AP 1.0, ML 3.5, DV -5.0; AP 0.6, ML 2.5, DV -4.5). Improvements of asymmetric motor behavior by the transplantation were examined every two weeks after the surgery. In two weeks, numbers of rotation by the experimental rats were (P<0.05) of the number before transplantation, however, the ratio increased slightly to in six weeks. In contrast, the ratio of sham-grafted animals ranged from 112.3+8.5% to 139.2+28.9% during the examination. Immunohistochemical studies further confirmed the presence, survival, migration, and expression of TH of the transplanted human cells.

Main strategy for a treatment of Parkinson's disease (PD), due to a progressive degeneration of dopaminergic neurons, is a pharmaceutical supplement of dopamine derivatives or ceil replacement therapy. Both of these protocols have pros and cons; former exhibiting a dramatic relief but causing a severe side effects on long-term prescription and latter also having a proven effectiveness but having availability and ethical problems Embryonic stem (ES) cells have several characteristics suitable for this purpose. To investigate a possibility of using ES cells as a carrier of therapeutic gene(s), human ES (hES, MB03) cells were transfected with cDNAs coding for tyrosine hydroxylase (TH) in pcDNA3.1 (+) and the transfectants were selected using neomycin (250 ). Expression of TH being confirmed, two of the positive clone (MBTH2 & 8) were second transfected with GTP cyclohydrolase 1 (GTPCH 1) in pcDNA3.1 (+)-hyg followed by selection with hygromycin-B (150 ) and RT-PCR confirmation. By immune-cytochemistry, these genetically modified but undifferentiated dual drug-resistant cells were found to express few of the neuronal markers, such as NF200, -tubulin, and MAP2 as well as astroglial marker GFAP. This results suggest that over-production of BH4 by ectopically expressed GTPCH I may be involved in the induction of those markers. Transplantation of the cells into striatum of 6-OHDA- denervated PD animal model relieved symptomatic rotational behaviors of the animals. Immunohistochemical analyses showed the presence of human cells within the striatum of the recipients. These results suggest a possibility of using hES cells as a carrier of therapeutic gene(s).