Focus on CRISPR: Transforming fat cells, may "lying thin" come true?

The number of obesity and metabolic syndrome is increasing rapidly around the world and will cause high morbidity and mortality. The medical community has been focusing on the development of prevention and treatment strategies for obesity and its complications. In mammals, both brown adipose tissue (BAT) and white adipose tissue (WAT) can promote energy homeostasis throughout the body, but their anatomical structures, shapes, and functions are quite different.
The activation of BAT increases energy expenditure, and its activity is inversely proportional to body mass index and fat mass, which makes BAT a promising anti-obesity therapeutic agent. BAT generates heat due to the expression of its unique uncoupling protein 1 (UCP1), which dissipates energy by uncoupling proton power from the production of adenosine triphosphate. Although the expression of UCP1 is limited to BAT under basal conditions, long-term cold exposure or β3-adrenergic stimulation can not only increase UCP1-mediated heat production in BAT, but also activate the accumulation of brown-like beige fat cells in WAT. In adults, WAT is distributed throughout the body and is located on the superficial fat pad; however, BAT exists in smaller areas of the deep fat pad, such as the cervix, supraclavicular, and paravertebral areas. Considering its richness and special location, WAT is easier to obtain and manipulate. WAT-induced browning may have great potential to prevent or treat obesity and obesity-related metabolic abnormalities.
Previous studies have shown that ectopic expression of UCP1 in mice can protect skeletal muscle and adipose tissue from diet-induced obesity. Pigs lack functional UCP1 gene, and ectopic expression of UCP1 in white fat promotes lipolysis. And cold resistance. These studies clearly demonstrated the anti-obesity effect of ectopic overexpressed UCP1 in animals. However, it is unclear whether these effects can be reproduced in humans by activating the endogenous UCP1 locus.
Autologous cell therapy is a preferred therapeutic intervention, in which cells are taken from an individual and administered to the same individual to minimize immune rejection. Autologous cell therapy has always been a very active research area, such as CRISPR. The CRISPR-Cas9 system provides a powerful means for genome editing in mammalian cells, and several new tools have been developed on the basis of CRISPR-Cas9 to achieve targeted inhibition or activation of gene expression. In CRISPR activation technology, nuclease-inactivated Cas9 is fused with the transactivation domain and guided by a single guide RNA targeting a specific promoter. This synthetic transcription complex can activate the expression of endogenous genes. The CRISPR activation system has been used to drive the differentiation, transdifferentiation and reprogramming of various mouse and human cell types. However, the effects of CRISPR-engineered cells on systemic metabolism and their therapeutic potential against obesity and obesity-related diseases have not been tested.
One of the advanced versions of CRISPR activation is the cooperative activation mediator (SAM) system, in which dCas9 binds to a fusion protein consisting of two transcriptional activation domains derived from the nuclear factor kappa light chain enhancer (NF-κB) of activated B cells Cooperating with heat shock factor 1 (HSF1) to promote transcription. Recently, researchers have used the CRISPR-SAM system in human white preadipocytes to activate UCP1 gene expression. In addition to characterizing these CRISPR-engineered human cells, the therapeutic potential of these cells is also demonstrated by treating obesity and metabolic disorders in mice. The research results were published in “Science Translational Medicine”.

Science|Focus on CRISPR: Transforming fat cells, “lying thin” may come true?
Endogenous activation of UCP1 by CRISPR-SAM triggers a brown-like phenotype in human white adipocytes
The researchers used the CRISPR-SAM system to increase the expression of endogenous UCP1 in human white preadipocytes, and designed four different guide RNAs to target about 50 to 150bp upstream of human UCP1 and a stable expression vector encoding dCas9 Fusion with four tandem repeats of viral protein transcription activator. The CRISPR-SAM system and lentiviruses of gRNA A to D were transduced into human white preadipocytes derived from two subjects. gRNA-A increases UCP1 mRNA by about 6000 times and UCP1 protein by about 20 times. These findings indicate that the combination of CRISPR-SAM targeting UCP1 and sgRNA can transform human white preadipocytes into human brown-like cells by turning on endogenous UCP1 expression.

Endogenous activation of UCP1 by CRISPR-SAM triggers a brown-like phenotype in human white adipocytes
Compared with primitive human white adipocytes lacking sgRNA, HUMBLE cells maintained higher UCP1 expression after adipogenic differentiation. However, the expression of UCP1 in HUMBLE cells was slightly lower than that of differentiated brown adipocytes of the same person. Compared with the white control adipocytes, HUMBLE cells showed elevated GLUT1 mRNA. Compared with brown control adipocytes, HUMBLE cells lead to increased mitochondrial activity and mitochondrial DNA content. Compared with white control adipocytes, HUMBLE cells showed a more slender and connected mitochondrial network, similar to the mitochondrial morphology of brown control adipocytes.
In addition to the molecular and structural characteristics of brown control fat cells, HUMBLE cells also acquired brown fat-like functional phenotypes. Compared with white control cells, when HUMBLE adipocytes use glucose as a substrate, their glucose uptake, basal respiration rate, proton leakage, and forskolin (FSK) dependent oxygen consumption rate (OCR) increase. Compared with the white control cells, they also have a higher fatty acid-dependent OCR ability, while the fatty acid intake has not changed. Using heat-sensitive fluorescent dyes, the researchers directly measured the heat produced by the cultured cells and found that compared with forskolin, the HUMBLE cells produced more heat than the white control cells. These findings indicate that HUMBLE cells have thermal capabilities in using glucose or fatty acids as fuel sources.

The transplantation of genetic engineering or pharmacologically induced beige/tan cells can improve the metabolic homeostasis in mice. In order to determine the metabolic effects of HUMBLE cells in vivo, the white control, HUMBLE and brown control pre-adipocytes were mixed with Matrigel, and then transplanted to the sternal area of immunocompromised nude mice. Two weeks after transplantation, HUMBLE cells showed the highest luciferase signal among the three transplanted cell lines. By the 4th week, a large amount of UCP1 expression was detected in both brown control cells and HUMBLE cells, which indicated that the pre-brown control adipocytes had differentiated into mature adipocytes, and UCP1 expression was up-regulated. In addition, as shown by the positive staining of mouse CD31 and tyrosine hydroxylase, respectively, the transplanted tissue became vascularized and innervated. The researchers detected human adiponectin in the serum of mice, indicating that these grafts can act as endocrine tissues to secrete adiponectin or other factors into the circulatory system. Taken together, these data indicate that transplanted pre-adipocytes can reconstruct adipose tissue in vivo, thereby recapitulating the phenotype of these cells in vitro.
To investigate whether HUMBLE cells can prevent DIO metabolic disorders in mice, the researchers monitored the metabolic phenotype of recipient mice that were fed a 45% high-fat diet (HFD) 2 weeks before transplantation. The mice that received HUMBLE and brown control adipocytes lost weight compared to mice that received white control cells. Compared with mice that received white control cells, the glucose tolerance and insulin sensitivity of mice transplanted with HUMBLE or brown control cells were also improved by about 30% to 35%. There was no statistically significant change in serum insulin. However, the concentration of circulating triglycerides was reduced in the HUMBLE and brown control cell groups compared to mice that received white control cells. Compared with mice receiving white control cell transplantation, brown control fat cell transplantation allowed the mice to maintain a higher core body temperature when exposed to cold.

To further verify the metabolism of HUMBLE cells in vitro and in vivo, the researchers used white preadipocytes isolated from another individual and the same CRISPR-SAM system to generate additional HUMBLE cell lines. Consistent with the above-mentioned protective effect on cells produced by donor A41, HUMBLE cells produced by donor A38 also show high thermogenesis potential in vitro, and their glucose tolerance and insulin sensitivity when transplanted in vivo using the same protocol Both sex and glucose tolerance are improved. These results indicate that the CRISPR-based HUMBLE cell strategy is applicable to different human subjects.

To examine the potential of using HUMBLE cells to treat obesity, the researchers transplanted the cells into DIO nude mice. The weight of DIO mice transplanted with HUMBLE or brown control cells is lighter than that of mice transplanted with white control cells, which is related to the significant improvement of glucose tolerance and insulin sensitivity and the decrease of circulating insulin concentration 4 weeks after transplantation. Compared with the mice transplanted with white control cells, the liver lipid content of mice transplanted with HUMBLE or brown control cells was reduced, and the lipid droplet size in endogenous BAT and subcutaneous WAT was correspondingly reduced. In conclusion, these data indicate that HUMBLE cell transplantation may be a potential anti-obesity treatment strategy.

Compared with mice with white control cell transplantation, mice that received HUMBLE or brown control cells cleared the glucose radiotracer faster from the circulation. It is not an increase in glucose uptake by transplanted cells. There was no change in endogenous scWAT or glucose uptake in skeletal muscle or the expression of any of the aforementioned genes. Functionally, compared with mice that received white control cells, mice that received HUMBLE or brown control cells had a higher surface temperature near endogenous BAT, suggesting the activation of adaptive thermogenesis in recipient mice. These data indicate that HUMBLE and brown control cells can activate endogenous BAT.

Results and discussion

In this study, the researchers used the CRISPR-SAM system to engineer human white preadipocytes to activate the endogenous UCP1 gene and drive a brown-like phenotype. By using paired human white and brown preadipocyte lines, it is possible to compare engineered HUMBLE cells and their syngeneic parent white control cells with true brown control cells of the same individual, thereby providing accurate phenotypic comparisons. Research data proves the preclinical therapeutic potential of CRISPR engineered HUMBLE cells in preventing and treating obesity. The transplantation of HUMBLE cells can greatly improve the glucose homeostasis in mice.
In the study, the transplanted human preadipocytes differentiated into adipocytes in situ. More importantly, in the obese mouse model, the vascularization and innervation required for adipocyte function were developed. In addition, the transplanted cells survived in the body for at least 12 weeks, so the beneficial effects of these cells on glucose homeostasis in mice continued. The results indicate that HUMBLE cell-based therapy can potentially be used to combat metabolic disorders caused by high-calorie diets.
This study has certain limitations. Researchers use immunocompromised nude mice as recipients of HUMBLE cell transplantation to avoid immune rejection of human cell transplantation. However, the lack of specific immune cells and cytokines in immunocompromised mice may affect the results and interpretation of metabolic evaluation. CRISPR, a gene editing tool called “magic shear”, has high hopes in genetic engineering applications. With its advantages of low cost, convenient operation and high efficiency, it has become a right-hand man in biological research, but CRISPR is undeniable. Currently facing problems and challenges such as easy off-target, Medicilon will continue to pay attention to the development of gene editing technology as a new drug research and development CRO, and look forward to the day when CRISPR technology matures and contributes to human health.