Stem Cell Clinic

Induced pluripotent stem cells and CRISPR reversed diabetes in mice – Drug Target Review

Induced pluripotent stem cells made to produce insulin and CRISPR, used to correct a genetic defect, cured Wolfram syndrome in mice.

Using induced pluripotent stem cells (iPSCs) produced from the skin of a patient with a rare, genetic form of insulin-dependent diabetes called Wolfram syndrome, researchers transformed the human stem cells into insulin-producing cells and used CRISPR-Cas9 to correct a genetic defect that had caused the syndrome. They then implanted the cells into lab mice and cured the unrelenting diabetes in those models.

The findings, from researchers at Washington University School of Medicine in St. Louis, US, suggest this CRISPR-Cas9 technique may hold promise as a treatment for diabetes, particularly the forms caused by a single gene mutation and it also may be useful one day in some patients with the more common forms of diabetes, such as type 1 and type 2.

This is the first time CRISPR has been used to fix a patients diabetes-causing genetic defect and successfully reverse diabetes, said co-senior investigator Dr Jeffrey Millman, an assistant professor of medicine and of biomedical engineering at Washington University. For this study, we used cells from a patient with Wolfram syndrome because, conceptually, we knew it would be easier to correct a defect caused by a single gene. But we see this as a stepping stone toward applying gene therapy to a broader population of patients with diabetes.

Wolfram syndrome is caused by mutations to a single gene, providing the researchers an opportunity to determine whether combining stem cell technology with CRISPR to correct the genetic error also might correct the diabetes caused by the mutation.

Researchers at Washington University School of Medicine in St. Louis have transformed stem cells into insulin-producing cells. They used the CRISPR gene-editing tool to correct a defect that caused a form of diabetes, and implanted the cells into mice to reverse diabetes in the animals. Shown is a microscopic image of insulin-secreting beta cells (insulin is green) that were made from stem cells produced from the skin of a patient with Wolfram syndrome [credit: Millman lab Washington University].

Millman and his colleagues had previously discovered how to convert human stem cells into pancreatic beta cells. When such cells encounter blood sugar, they secrete insulin. Recently, these researchers developed a new technique to more efficiently convert human stem cells into beta cells that are considerably better at controlling blood sugar.

In this study, they took the additional steps of deriving these cells from patients and using the CRISPR-Cas9 gene-editing tool on those cells to correct a mutation to the gene that causes Wolfram syndrome (WFS1). Then, the researchers compared the gene-edited cells to insulin-secreting beta cells from the same batch of stem cells that had not undergone editing with CRISPR.

In the test tube and in mice with a severe form of diabetes, the newly grown beta cells that were edited with CRISPR more efficiently secreted insulin in response to glucose. Diabetes disappeared in mice with the CRISPR-edited cells implanted beneath the skin and the animals blood sugar levels remained in normal range for the entire six months they were monitored. Animals receiving unedited beta cells remained diabetic. Although their newly implanted beta cells could produce insulin, it was not enough to reverse their diabetes.

We basically were able to use these cells to cure the problem, making normal beta cells by correcting this mutation, said co-senior investigator Dr Fumihiko Urano, the Samuel E. Schechter Professor of Medicine and a professor of pathology and immunology. Its a proof of concept demonstrating that correcting gene defects that cause or contribute to diabetes in this case, in the Wolfram syndrome gene we can make beta cells that more effectively control blood sugar. Its also possible that by correcting the genetic defects in these cells, we may correct other problems Wolfram syndrome patients experience, such as visual impairment and neurodegeneration.

Were excited about the fact that we were able to combine these two technologies growing beta cells from induced pluripotent stem cells and using CRISPR to correct genetic defects, Millman said. In fact, we found that corrected beta cells were indistinguishable from beta cells made from the stem cells of healthy people without diabetes.

Moving forward, the process of making beta cells from stem cells should get easier, the researchers said. For example, the scientists have developed less intrusive methods, making iPSCs from blood and they are working on developing stem cells from urine samples.

The study is published in Science Translational Medicine.

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Induced pluripotent stem cells and CRISPR reversed diabetes in mice - Drug Target Review

Reversing diabetes with CRISPR and patient-derived stem cells – FierceBiotech

Insulin injections cancontrol diabetes, but patients still experience serious complications such as kidney disease and skin infections. Transplanting pancreatic tissues containing functional insulin-producing beta cells is of limited use, because donors are scarce and patients must take immunosuppressant drugs afterward.

Now, scientists atWashington University in St. Louis havedeveloped a way to use gene editing system CRISPR-Cas9 to edit a mutation in human-induced pluripotent stem cells (iPSCs) and then turnthem into beta cells. When transplanted into mice, the cells reversed preexisting diabetes in a lasting way, according to results published in the journal Science Translational Medicine.

While the researchers used cells from patients with Wolfram syndromea rare childhood diabetes caused by mutations in the WFS1 genethey argue that the combination of a gene therapy with stem cells could potentially treat other forms of diabetes as well.

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One of the biggest challenges we faced was differentiating our patient cells into beta cells. Previous approaches do not allow for this robust differentiation. We use our new differentiation protocol targeting different development and signaling pathways to generate our cells, the studys lead author, Kristina Maxwell, explained in a video statement.

Making pancreatic beta cells from patient-derived stem cells requires precise activation and repression of specific pathways, and atthe right times, to guide the development process. In a recent Nature Biotechnology study, the team described a successful method that leverages the link between a complex known as actin cytoskeleton and the expression of transcription factors that drive pancreatic cell differentiation.

This time, the researchers applied the technology to iPSCs from two patients with Wolfram syndrome. They used CRISPR to correct the mutated WFS1 gene in the cells and differentiated the edited iPSCs into fully functional beta cells.

After transplanting the corrected beta cells into diabetic mice, the animals saw their blood glucose drop quickly, suggesting the disease had been reversed. The effect lasted for the entire six-month observation period, the scientists reported. By comparison, those receiving unedited cells from patients were unable to achieve glycemic control.

RELATED:CRISPR Therapeutics, ViaCyte team up on gene-edited diabetes treatment

The idea of editing stem cells with CRISPR has already attracted interest in the biopharma industry. Back in 2018, CRISPR Therapeutics penned a deal with ViaCyte to develop off-the-shelf, gene-editing stem cell therapies for diabetes. Rather than editing iPSCs from particular patients themselves to correct a faulty gene, the pairs lead project used CRISPR to edit healthy cells so that they lackedthe B2M gene and expressed PD-L1 to protect against immune attack. The two companies unveiled positive preclinical data inSeptember.

Other research groups working on gene therapy or stem cells for diabetes include a Harvard University scientist and his startup Semma Therapeutics, whichdeveloped a method for selecting beta cells out of a mixture of cells developed from PSCs. Scientists at the University of Wisconsin-Madison recently proposed that removing the IRE1-alpha gene in beta cells could prevent immune T cells from attacking them in mice with Type 1 diabetes.

The Washington University team hopes its technology may help Type 1 diabetes patients whose disease is caused by multiple genetic and environmental factors as well as the Type 2 form linked to obesity and insulin resistance.

We can generate a virtually unlimited number of beta cells from patients with diabetes to test and discover new drugs to hopefully stop or even reverse this disease, Jeffrey Millman, the studys co-senior author, said in the video statement. Perhaps most importantly, this technology now allows for the potential use of gene therapy in combination with the patients own cells to treat their own diabetes by transplantation of lab-grown beta cells.

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Reversing diabetes with CRISPR and patient-derived stem cells - FierceBiotech

The integrated stress response: From mechanism to disease – Science Magazine

Proteostasis dISRupted

Despite their importance, many crucial networks for protein quality control within cells diminish with age. The resulting loss of proteostasis, the process by which the health of a cell's proteins is monitored and maintained, is associated with a wide range of age-related human diseases. Costa-Mattioli and Walter review the integrated stress response (ISR), a central signaling network that responds to proteostasis defects by tuning protein synthesis. The ISR is activated in a wide range of pathological conditions, so a mechanistic understanding of its pathway may help in the development of therapeutic tools through which it can be modulated.

Science, this issue p. eaat5314

The integrated stress response (ISR) is an evolutionarily conserved intracellular signaling network that helps the cell, tissue, and organism to adapt to a variable environment and maintain health. In response to different environmental and pathological conditions, including protein homeostasis (proteostasis) defects, nutrient deprivation, viral infection, and oxidative stress, the ISR restores balance by reprogramming gene expression. The various stresses are sensed by four specialized kinases (PERK, GCN2, PKR and HRI) that converge on phosphorylation of a single serine on the eukaryotic translation initiation factor eIF2. eIF2 phosphorylation blocks the action of eIF2s guanine nucleotide exchange factor termed eIF2B, resulting in a general reduction in protein synthesis. Paradoxically, phosphorylation of eIF2 also triggers the translation of specific mRNAs, including key transcription factors, such as ATF4. These mRNAs contain short inhibitory upstream open reading frames in their 5-untranslated regions that prevent translation initiation at their canonical AUGs. By tuning down general mRNA translation and up-regulating the synthesis of a few proteins that drive a new transcriptional program, the ISR aims to maintain or reestablish physiological homeostasis. However, if the stress cannot be mitigated, the ISR triggers apoptosis to eliminate the damaged cell.

Our understanding of the central mechanisms that govern the ISR has advanced vastly. The ISRs central regulatory hub lies in the eIF2eIF2B complex, which controls the formation of the eIF2GTPmethionyl-intiator tRNA ternary complex (TC), a prerequisite for initiating new protein synthesis. Assembly of functional TC is inhibited by eIF2-P, which blocks eIF2B noncompetitively. In mammalian cells, the phosphorylation of eIF2 is a tightly regulated process. In addition to the four specialized eIF2 kinases that phosphorylate eIF2, two dedicated phosphatases antagonize this reaction. Both phosphatases contain a common catalytic core subunit, the protein phosphatase 1 (PP1), and a regulatory subunit (GADD34 or CReP), which render the phosphatase specific to eIF2. Structural and biophysical approaches have elucidated the mechanism of action of eIF2B and its modulation by ISR inhibitors and activators. Gene expression analyses have revealed complex ISR-driven reprogramming. Although it has been long recognized that, in the brain, long-term memory formation requires new protein synthesis, recent causal and convergent evidence across different species and model systems has shown that the ISR serves as a universal regulator of this process. Briefly, inhibition of the ISR enhances long-term memory formation, whereas activation of the ISR prevents it. Consistent with this notion, unbiased genome-wide association studies have identified mutations in key components of the ISR in humans with intellectual disability. Furthermore, age-related cognitive disorders are commonly associated with the activation of the ISR. Most notably, oxidative stress, misfolded proteins, and other stressors induce the ISR in several neurodegenerative disorders, including Alzheimers disease. Recent genetic and pharmacological evidence suggest that tuning the ISR reverses cognitive dysfunction as well as neurodegeneration in a wide range of memory disorders that result from protein homeostasis defects. Thus, long-term memory deficits may primarily result as a consequence of ISR activation rather than from the particular proteostasis defects that lead to its induction. Finally, the ISR is also implicated in the pathogenesis of a plethora of other complex diseases, including cancer, diabetes, and metabolic disorders.

The ISR is emerging as a central regulator of protein homeostasis at both the cellular and organismal level. Mechanistically, much remains to be understood regarding additional inputs into the eIF2BeIF2 regulatory hub controlling TC concentration, as well as the IRSs connectivity to other intracellular signaling networks. As yet, little is known about the role of the specific proteins whose synthesis is altered during acute and persistent ISR activation and how these effectors collaborate to compute the life or death decisions cells make upon ISR activation. ISR gene expression signatures and functional consequences will need to be mapped across different tissues, cell types, and developmental stages. In addition, it will be invaluable to generate additional genetic and molecular tools that permit the direct temporal and spatial manipulation of ISR pathway in specific cells and circuits to determine their function. From a medical perspective, the ISR is implicated in the etiology of several disorders, and manipulation of the ISR is emerging as a promising therapeutic avenue for the treatment of a variety of diseases. The use of innovative mouse models, patient-derived induced pluripotent stem cells, and human organoids will greatly enhance our ability to explore the ISRs clinical relevance further and help define therapeutic windows in which ISR modulation may prove beneficial. Identifying additional specific small-molecule inhibitors and activators of the ISR will offer valuable opportunities to dissect the role of the ISR pharmacologically in health and disease. Finally, discovery and mechanistic understanding of additional ISR modulators will increase the repertoire of therapeutic targets and may further enable clinical development in a wide range of age-related human diseases.

Diverse deviations from homeostasis activate the ISR. The resulting dysregulation of translation contributes to numerous diseases.

Protein quality control is essential for the proper function of cells and the organisms that they make up. The resulting loss of proteostasis, the processes by which the health of the cells proteins is monitored and maintained at homeostasis, is associated with a wide range of age-related human diseases. Here, we highlight how the integrated stress response (ISR), a central signaling network that responds to proteostasis defects by tuning protein synthesis rates, impedes the formation of long-term memory. In addition, we address how dysregulated ISR signaling contributes to the pathogenesis of complex diseases, including cognitive disorders, neurodegeneration, cancer, diabetes, and metabolic disorders. The development of tools through which the ISR can be modulated promises to uncover new avenues to diminish pathologies resulting from it for clinical benefit.

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The integrated stress response: From mechanism to disease - Science Magazine