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CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?

ABSTRACT: Due to their ease of isolation, differentiation capabilities, and immunomodulatory properties, the therapeutic potential of mesenchymal stem cells (MSCs) has been assessed in numerous pre-clinical and clinical settings. Currently, whole pancreas or islet transplantation is the only cure for people with type 1 diabetes (T1D) and, due to the autoimmune nature of the disease, MSCs have been utilised either natively or transdifferentiated into insulin-producing cells (IPCs) as an alternative treatment. However, the initial success in pre-clinical animal models has not translated into successful clinical outcomes. Thus, this review will summarise the current state of MSC-derived therapies for the treatment of T1D in both the pre-clinical and clinical setting, in particular their use as an immunomodulatory therapy and targets for the generation of IPCs via gene modification. In this review, we highlight the limitations of current clinical trials of MSCs for the treatment of T1D, and suggest the novel clustered regularly interspaced short palindromic repeat (CRISPR) gene-editing technology and improved clinical trial design as strategies to translate pre-clinical success to the clinical setting.

SUBMITTER: Gerace D

PROVIDER: S-EPMC5345178 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?

Gerace Dario D Martiniello-Wilks Rosetta R Nassif Najah Therese NT Lal Sara S Steptoe Raymond R Simpson Ann Margaret AM

Stem cell research & therapy 20170309 1

Due to their ease of isolation, differentiation capabilities, and immunomodulatory properties, the therapeutic potential of mesenchymal stem cells (MSCs) has been assessed in numerous pre-clinical and clinical settings. Currently, whole pancreas or islet transplantation is the only cure for people with type 1 diabetes (T1D) and, due to the autoimmune nature of the disease, MSCs have been utilised either natively or transdifferentiated into insulin-producing cells (IPCs) as an alternative treatme ...[more]

PMID: 28279194

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Project description:Background & aimsWilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by loss-of-function mutations in ATP7B, which encodes a copper-transporting protein. It is characterized by excessive copper deposition in tissues, predominantly in the liver and brain. We sought to investigate whether gene-corrected patient-specific induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) could serve as an autologous cell source for cellular transplantation therapy in WD.MethodsWe first compared the in vitro phenotype and cellular function of ATP7B before and after gene correction using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) in iHeps (derived from patients with WD) which were homozygous for the ATP7B R778L mutation (ATP7BR778L/R778L). Next, we evaluated the in vivo therapeutic potential of cellular transplantation of WD gene-corrected iHeps in an immunodeficient WD mouse model (Atp7b -/- / Rag2 -/- / Il2rg -/- ; ARG).ResultsWe successfully created iPSCs with heterozygous gene correction carrying 1 allele of the wild-type ATP7B gene (ATP7BWT/-) using CRISPR/Cas9 and ssODNs. Compared with ATP7BR778L/R778L iHeps, gene-corrected ATP7BWT/- iHeps restored i n vitro ATP7B subcellular localization, its subcellular trafficking in response to copper overload and its copper exportation function. Moreover, in vivo cellular transplantation of ATP7BWT/- iHeps into ARG mice via intra-splenic injection significantly attenuated the hepatic manifestations of WD. Liver function improved and liver fibrosis decreased due to reductions in hepatic copper accumulation and consequently copper-induced hepatocyte toxicity.ConclusionsOur findings demonstrate that gene-corrected patient-specific iPSC-derived iHeps can rescue the in vitro and in vivo disease phenotypes of WD. These proof-of-principle data suggest that iHeps derived from gene-corrected WD iPSCs have potential use as an autologous ex vivo cell source for in vivo therapy of WD as well as other inherited liver disorders.Lay summaryGene correction restored ATP7B function in hepatocytes derived from induced pluripotent stem cells that originated from a patient with Wilson's disease. These gene-corrected hepatocytes are potential cell sources for autologous cell therapy in patients with Wilson's disease.

Project description:UnlabelledThe clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3'?5' exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters.ImportanceRecent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways.

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CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?

Publications

CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?

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