scholarly journals Co-opting regulation bypass repair (CRBR) as a gene correction strategy for monogenic diseases

2020 ◽  
Author(s):  
Jingjie Hu ◽  
Rebecca A. Bourne ◽  
Barbara C. McGrath ◽  
Alice Lin ◽  
Zifei Pei ◽  
...  
Keyword(s):  
Genetic Defect ◽  
Regulatory Region ◽  
Neonatal Diabetes ◽  
Large Deletion ◽  
Gene Repair ◽  
Gene Correction ◽  
Pancreatic Islets Of Langerhans ◽  
Insulin Promoter ◽  
Monogenic Diseases ◽  
Mitotic Cells

With the development of CRISPR/Cas9-mediated gene editing technologies, correction of disease- causing mutations has become possible. However, current gene correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene correction strategy, Co-opting Regulation Bypass Repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end-joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR gene repair results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We demonstrated the potential of CRBR strategy for human gene therapy by rescuing a mouse model of the Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either large deletion or nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we expressed a GFP CDS-terminator cassette that was integrated downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans which paves the way for autologous cell-tissue replacement therapy for gene repair in beta cells.

scholarly journals The Krüppel-Like Protein Gli-Similar 3 (Glis3) Functions as a Key Regulator of Insulin Transcription

2013 ◽  
Vol 27 (10) ◽  
pp. 1692-1705 ◽  
Author(s):  
Gary T. ZeRuth ◽  
Yukimasa Takeda ◽  
Anton M. Jetten
Keyword(s):  
Transcriptional Regulation ◽  
Regulatory Region ◽  
Neonatal Diabetes ◽  
Creb Binding Protein ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Synergistic Activation ◽  
Transcriptional Regulatory ◽  
Insulin Promoter ◽  
Regulatory Complex

Transcriptional regulation of insulin in pancreatic β-cells is mediated primarily through enhancer elements located within the 5′ upstream regulatory region of the preproinsulin gene. Recently, the Krüppel-like transcription factor, Gli-similar 3 (Glis3), was shown to bind the insulin (INS) promoter and positively influence insulin transcription. In this report, we examined in detail the synergistic activation of insulin transcription by Glis3 with coregulators, CREB-binding protein (CBP)/p300, pancreatic and duodenal homeobox 1 (Pdx1), neuronal differentiation 1 (NeuroD1), and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA). Our data show that Glis3 expression, the binding of Glis3 to GlisBS, and its recruitment of CBP are required for optimal activation of the insulin promoter in pancreatic β-cells not only by Glis3, but also by Pdx1, MafA, and NeuroD1. Mutations in the GlisBS or small interfering RNA−directed knockdown of GLIS3 diminished insulin promoter activation by Pdx1, NeuroD1, and MafA, and neither Pdx1 nor MafA was able to stably associate with the insulin promoter when the GlisBS were mutated. In addition, a GlisBS mutation in the INS promoter implicated in the development of neonatal diabetes similarly abated activation by Pdx1, NeuroD1, and MafA that could be reversed by increased expression of exogenous Glis3. We therefore propose that recruitment of CBP/p300 by Glis3 provides a scaffold for the formation of a larger transcriptional regulatory complex that stabilizes the binding of Pdx1, NeuroD1, and MafA complexes to their respective binding sites within the insulin promoter. Taken together, these results indicate that Glis3 plays a pivotal role in the transcriptional regulation of insulin and may serve as an important therapeutic target for the treatment of diabetes.

scholarly journals Eliminating SCID row: new approaches to SCID

Hematology ◽  
2014 ◽  
Vol 2014 (1) ◽  
pp. 475-480 ◽  
Author(s):  
Donald B. Kohn
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Ex Vivo ◽  
Autologous Transplantation ◽  
Unrelated Donor ◽  
Primary Immune Deficiency ◽  
Gene Repair ◽  
Gene Correction ◽  
Hematopoietic Stem ◽  
New Approaches

Abstract Treatments for patients with SCID by hematopoietic stem cell transplantation (HSCT) have changed this otherwise lethal primary immune deficiency disorder into one with an increasingly good prognosis. SCID has been the paradigm disorder supporting many key advances in the field of HSCT, with first-in-human successes with matched sibling, haploidentical, and matched unrelated donor allogeneic transplantations. Nevertheless, the optimal approaches for HSCT are still being defined, including determining the optimal stem cell sources, the use and types of pretransplantation conditioning, and applications for SCID subtypes associated with radiosensitivity, for patients with active viral infections and for neonates. Alternatively, autologous transplantation after ex vivo gene correction (gene therapy) has been applied successfully to the treatment of adenosine deaminase–deficient SCID and X-linked SCID by vector-mediated gene addition. Gene therapy holds the prospect of avoiding risks of GVHD and would allow each patient to be their own donor. New approaches to gene therapy by gene correction in autologous HSCs using site-specific endonuclease-mediated homology-driven gene repair are under development. With newborn screening becoming more widely adopted to detect SCID patients before they develop complications, the prognosis for SCID is expected to improve further. This chapter reviews recent advances and ongoing controversies in allogeneic and autologous HSCT for SCID.

scholarly journals Diabetes induced by gain-of-function mutations in the Kir6.1 subunit of the KATP channel

2016 ◽  
Vol 149 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Maria S. Remedi ◽  
Jonathan B. Friedman ◽  
Colin G. Nichols
Keyword(s):  
Insulin Secretion ◽  
Katp Channel ◽  
Vascular System ◽  
Wild Type Mouse ◽  
Neonatal Diabetes ◽  
Katp Channels ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Gain Of Function ◽  
Insulin Promoter

Gain-of-function (GOF) mutations in the pore-forming (Kir6.2) and regulatory (SUR1) subunits of KATP channels have been identified as the most common cause of human neonatal diabetes mellitus. The critical effect of these mutations is confirmed in mice expressing Kir6.2-GOF mutations in pancreatic β cells. A second KATP channel pore-forming subunit, Kir6.1, was originally cloned from the pancreas. Although the prominence of this subunit in the vascular system is well documented, a potential role in pancreatic β cells has not been considered. Here, we show that mice expressing Kir6.1-GOF mutations (Kir6.1[G343D] or Kir6.1[G343D,Q53R]) in pancreatic β cells (under rat-insulin-promoter [Rip] control) develop glucose intolerance and diabetes caused by reduced insulin secretion. We also generated transgenic mice in which a bacterial artificial chromosome (BAC) containing Kir6.1[G343D] is incorporated such that the transgene is only expressed in tissues where Kir6.1 is normally present. Strikingly, BAC-Kir6.1[G343D] mice also show impaired glucose tolerance, as well as reduced glucose- and sulfonylurea-dependent insulin secretion. However, the response to K+ depolarization is intact in Kir6.1-GOF mice compared with control islets. The presence of native Kir6.1 transcripts was demonstrated in both human and wild-type mouse islets using quantitative real-time PCR. Together, these results implicate the incorporation of native Kir6.1 subunits into pancreatic KATP channels and a contributory role for these subunits in the control of insulin secretion.

P061 Type 3 von Willebrand disease in Hungary: A partial large deletion is the most common genetic defect

Blood Reviews ◽  
2007 ◽  
Vol 21 ◽  
pp. S105
Author(s):  
A. Mohl ◽  
Z. Boda ◽  
R. Jager ◽  
H. Losonczy ◽  
A. Marosi ◽  
...  
Keyword(s):  
Genetic Defect ◽  
Large Deletion ◽  
Von Willebrand Disease ◽  
Type 3 ◽  
Von Willebrand

The insulin gene contains multiple transcriptional elements that respond to glucose

1994 ◽  
Vol 14 (6) ◽  
pp. 4067-4075
Author(s):  
M S German ◽  
J Wang
Keyword(s):  
Beta Cells ◽  
Transcriptional Response ◽  
Chloramphenicol Acetyltransferase ◽  
Insulin Gene ◽  
Single Mutation ◽  
Multiple Sequence ◽  
Glucose Response ◽  
Nuclear Extracts ◽  
Pancreatic Islets Of Langerhans ◽  
Insulin Promoter

The beta cells in the pancreatic islets of Langerhans increase insulin gene transcription in response to increased glucose concentration. We have mapped sequences within the rat insulin I gene 5'-flanking DNA (rInsI promoter) that direct this transcriptional response to glucose. When linked to chloramphenicol acetyltransferase and expressed in cultured beta cells, no single mutation of the rInsI promoter removes its ability to respond to glucose, although several mutations cause marked reductions in basal chloramphenicol acetyltransferase expression. A 50-bp sequence isolated from the rInsI promoter, the Far-FLAT minienhancer, can confer glucose responsiveness to nonresponsive promoters. Fine mapping of this minienhancer further localizes a glucose response to the sequence GGCCATCTGGCC, or the Far element. Nuclear extracts from islets grown in various glucose concentrations demonstrate a glucose-stimulated increase in a protein complex that binds the Far element and contains the transcription factors Pan-1 and Pan-2. Overexpression of intact or partially deleted Pan-1 ablates the Far-directed transcriptional response to glucose. We conclude that the full glucose response of the insulin promoter involves the interaction of multiple sequence elements. Part of this response, however, results from activation of a complex binding at the Far element.

The personalized approach to neonatal diabetes therapy depending on the genetic defect

2017 ◽  
Vol 1 (1) ◽  
pp. 36-39 ◽  
Author(s):  
Yulia V. Tikhonovich ◽  
Natalia A. Zubkova ◽  
Anatoly N. Tiulpakov
Keyword(s):  
Katp Channel ◽  
Genetic Disorders ◽  
Genetic Defect ◽  
Clinical Importance ◽  
Neonatal Diabetes ◽  
Diabetes Treatment ◽  
Diabetes Therapy ◽  
Common Causes ◽  
Genetic Examination ◽  
Personalized Approach

Neonatal diabetes mellitus (NDM) is defined as a heterogeneous group of genetic disorders with onset before 6 months of age. Mutations in KATP channel genes (KCNJ11, ABCC8) and the insulin gene (INS) are the most common causes of NDM. Accurate molecular diagnosis of NDM has significant clinical importance as it may influence diabetes treatment, explain pleiotropic features and define the prognosis in the examined subject as well as in other family members . In this report we present the results of a genetic examination of 70 patients with NDM, generalized the experience of using sulfonylurea in patients with KCNJ11 and ABCC8 genes mutations for the period from 2009 to 2016. A correlation is shown between the type of mutation, the course of the disease, and the sensitivity of patients to glibenclamide.

scholarly journals High-Frequency Intermolecular Homologous Recombination during Herpes Simplex Virus-Mediated Plasmid DNA Replication

2002 ◽  
Vol 76 (12) ◽  
pp. 5866-5874 ◽  
Author(s):  
Xinping Fu ◽  
Hua Wang ◽  
Xiaoliu Zhang
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Homologous Recombination ◽  
Single Molecule ◽  
High Frequency ◽  
Simian Virus 40 ◽  
Gene Repair ◽  
Gene Correction ◽  
Simplex Virus

ABSTRACT Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.

scholarly journals Chemotaxis to cAMP and slug migration in Dictyostelium both depend on migA, a BTB protein.

1997 ◽  
Vol 8 (9) ◽  
pp. 1763-1775 ◽  
Author(s):  
R Escalante ◽  
D Wessels ◽  
D R Soll ◽  
W F Loomis
Keyword(s):  
Mutant Strain ◽  
Restriction Enzyme ◽  
Protein Interactions ◽  
Actin Polymerization ◽  
Genetic Defect ◽  
Regulatory Region ◽  
Receptor Occupancy ◽  
Full Length ◽  
Developmental Defects ◽  
Chemical Gradients

Chemotaxis in natural aggregation territories and in a chamber with an imposed gradient of cyclic AMP (cAMP) was found to be defective in a mutant strain of Dictyostelium discoideum that forms slugs unable to migrate. This strain was selected from a population of cells mutagenized by random insertion of plasmids facilitated by introduction of restriction enzyme (a method termed restriction enzyme-mediated integration). We picked this strain because it formed small misshapen fruiting bodies. After isolation of portions of the gene as regions flanking the inserted plasmid, we were able to regenerate the original genetic defect in a fresh host and show that it is responsible for the developmental defects. Transformation of this recapitulated mutant strain with a construct carrying the full-length migA gene and its upstream regulatory region rescued the defects. The sequence of the full-length gene revealed that it encodes a novel protein with a BTB domain near the N terminus that may be involved in protein-protein interactions. The migA gene is expressed at low levels in all cells during aggregation and then appears to be restricted to prestalk cells as a consequence of rapid turnover in prespore cells. Although migA- cells have a dramatically reduced chemotactic index to cAMP and an abnormal pattern of aggregation in natural waves of cAMP, they are completely normal in size, shape, and ability to translocate in the absence of any chemotactic signal. They respond behaviorally to the rapid addition of high levels of cAMP in a manner indicative of intact circuitry connecting receptor occupancy to restructuring of the cytoskeleton. Actin polymerization in response to cAMP is also normal in the mutant cells. The defects at both the aggregation and slug stage are cell autonomous. The MigA protein therefore is necessary for efficiently assessing chemical gradients, and its absence results in defective chemotaxis and slug migration.

scholarly journals Co-opting regulation bypass repair as a gene correction strategy for monogenic diseases

2021 ◽  
Author(s):  
Jingjie Hu ◽  
Rebecca A. Bourne ◽  
Barbara C. McGrath ◽  
Alice Lin ◽  
Zifei Pei ◽  
...  
Keyword(s):  
Gene Correction ◽  
Monogenic Diseases

scholarly journals Gene Correction for SCID-X1 in Long-Term Hematopoietic Stem Cells

2018 ◽  
Author(s):  
Mara Pavel-Dinu ◽  
Volker Wiebking ◽  
Beruh T. Dejene ◽  
Waracharee Srifa ◽  
Sruthi Mantri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genome Editing ◽  
Limit Of Detection ◽  
Severe Combined Immunodeficiency ◽  
Gene Correction ◽  
Hematopoietic Stem ◽  
High Frequencies ◽  
Monogenic Diseases

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. High frequencies of reproducible targeted integration of a wild-type cDNA into the endogenous start codon of a gene in LT-HSCs could provide a robust genome editing approach to cure genetic diseases in which patients have different mutations throughout the gene. We describe a clinically relevant method for correcting X-linked severe combined immunodeficiency (SCID-X1). By using a highly specific and active CRISPR/Cas9-AAV6 based strategy and selection-free approach, we achieve up to 20% genome integration frequencies in LT-HSCs of a full-length IL2RG cDNA at the endogenous start site as demonstrated by serial transplantation and analysis of genome edited human cells eight months following initial transplantation. In addition to high frequencies of functional gene correction in LT-HSCs we observed no evidence of abnormal hematopoiesis following transplantation, a functional measure of the lack of genotoxicity. Deep analysis of potential off-target activity detected two sites with low frequency (<0.3%) of off-target mutations. The level of off-target mutations was reduced to below the limit of detection using a high fidelity Cas9. Moreover, karyotype evaluation identified no genomic instability events. We achieved high levels of genome targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect of patient derived cells both in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.

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