Even with treatment, patients experience complications, as current therapies have limited effectiveness, especially with delayed initiation. Because the pathology of the disease begins before birth, the research team saw the syndrome as a candidate for prenatal treatment. With this goal, the researchers used CRISPR base editing, which requires only a single stranded DNA break and is thought to be more efficient and safer than other editing approaches, to convert the mutated adenine to guanine in the mouse model of MPS-IH.
The researchers used an adeno-associated virus serotype 9 AAV9 viral vector to deliver the base editor to a fetal mouse model. They showed that prenatally treated mice demonstrated increased survival and improvement of metabolic, skeletal, and cardiac disease. Of note, the researchers observed corrected cells not only in the liver, but also in the heart, demonstrating the treatment was effective in multiple organs.
To assess the feasibility of the treatment after birth, the researchers tested the approach in week-old MPS-IH mice and observed efficient on-target editing in the heart and liver, which was also associated with cardiac improvement. Whereas control models with the disease experienced cardiac decline between 4 and 6 months, sometimes resulting in death, postnatally treated mice demonstrated cardiac disease at 4 months, but the progression slowed between 4 and 6 months of age.
Similar to prenatal treated mice, none of the mice treated after birth died by the study endpoint. Bose et al. Its pediatric research program is among the largest in the country. In addition, its unique family-centered care and public service programs have brought the bed hospital recognition as a leading advocate for children and adolescents. To counteract this virulence factor, host cells initiate receptor-interacting serine-threonine protein kinase 1 RIPK1 —dependent caspase-8—directed gasdermin D GSDMD cleavage and inflammatory cell death pyroptosis when TAK1 is inhibited.
Inflammatory caspase cleavage of GSDMD causes cell membrane pores that mediate both pyroptosis and proinflammatory cytokine secretion. An alternate pyroptotic pathway, mediated by activation of RIPK1 and caspase-8, is triggered when the YopJ virulence factor secreted during pathogenic Yersinia infection blocks TAK1 activation.
To determine the molecular mechanisms underlying Yersinia activation of RIPK1—caspase-8—dependent pyroptosis, we performed a genome-wide CRISPR screen using Cas9-expressing immortalized mouse bone marrow—derived macrophages infected with a genome-wide library of single-guide RNA—encoding lentiviruses.
The genomes of cells resistant to caspase-8— or caspase—dependent pyroptosis were sequenced to identify the knocked-out genes required for pyroptosis. The screen identified multiple genes in the lysosomal membrane—anchored Folliculin Flcn —Folliculin-interacting protein 2 Fnip2 —Rag-Ragulator complex as necessary for caspase-8— but not caspase—mediated pyroptosis.
Deficiency of Rag-Ragulator complex genes rendered cells highly resistant to TAK1 inhibition—triggered pyroptosis, indicating a critical and unexpected role of the lysosomal membrane—tethered Rag-Ragulator supercomplex in RIPK1-dependent caspase-8—directed pyroptosis.
Activation of RIPK1 phosphorylation, caspase-8 activation, and pyroptosis depended on Rag guanosine triphosphatase GTPase activity and Rag-Ragulator lysosomal binding but was independent of the mechanistic target of rapamycin complex 1 mTORC1 , a well-known Rag-Ragulator—regulated complex.
By contrast, Rag-Ragulator did not regulate canonical or noncanonical inflammasome-triggered pyroptosis. Our study revealed an instructive role of metabolic signaling in directing TAK1 inhibition—induced pyroptosis during a pathogenic bacterial infection. Rag-Ragulator is a well-known critical regulator of cellular responses to changes in nutrient availability and metabolism.
Rag GTPase activity was critical for triggering the pathway. The role found here for Rag-Ragulator in pyroptosis expands its known roles in metabolic regulation to include regulation of the response to pathogenic infection. Rag-Ragulator monitors both metabolism and infection to serve as a central hub for helping to decide whether available nutrients are adequate for cell proliferation and if an infected cell should die and send out inflammatory danger signals.
Future studies can further explore the conditions that stimulate caspase-8—mediated pyroptosis and provide more mechanistic details of how it is regulated, as well as investigate whether manipulating this pathway could have therapeutic benefit.
Host cells initiate cell death programs to limit pathogen infection. Thus, the lysosomal metabolic regulator Rag-Ragulator instructs the inflammatory response to Yersinia.
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