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Article ; Online: Revertant Mosaicism in Epidermolysis Bullosa

Cameron Meyer-Mueller / Mark J. Osborn / Jakub Tolar / Christina Boull / Christen L. Ebens

Biomedicines, Vol 10, Iss 114, p

2022 Volume 114

Abstract: Epidermolysis bullosa (EB) is a group of genetic blistering diseases characterized by mechanically fragile skin and mucocutaneous involvement. Historically, disease management has focused on supportive care. The development of new genetic, cellular, and ... ...

Abstract	Epidermolysis bullosa (EB) is a group of genetic blistering diseases characterized by mechanically fragile skin and mucocutaneous involvement. Historically, disease management has focused on supportive care. The development of new genetic, cellular, and recombinant protein therapies has shown promise, and this review summarizes a unique gene and cell therapy phenomenon termed revertant mosaicism (RM). RM is the spontaneous correction of a disease-causing mutation. It has been reported in most EB subtypes, some with relatively high frequency, and has been observed in both keratinocytes and fibroblasts. RM manifests as identifiable patches of unaffected, blister-resistant skin and can occur through a variety of molecular mechanisms, including true back mutation, intragenic crossover, mitotic gene conversion, and second-site mutation. RM cells represent a powerful autologous platform for therapy, and leveraging RM cells as a therapeutic substrate may avoid the inherent mutational risks of gene therapy/editing. However, further examination of the genomic integrity and long-term functionality of RM-derived cells, as well in vivo testing of systemic therapies with RM cells, is required to realize the full therapeutic promise of naturally occurring RM in EB.
Keywords	epidermolysis bullosa ; revertant mosaicism ; cellular therapy ; gene therapy ; autograft ; loss of heterozygosity ; Biology (General) ; QH301-705.5
Subject code	610
Language	English
Publishing date	2022-01-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production

Colby J. Feser / Christopher J. Lees / Daniel T. Lammers / Megan J. Riddle / Jason R. Bingham / Matthew J. Eckert / Jakub Tolar / Mark J. Osborn

International Journal of Molecular Sciences, Vol 23, Iss 5090, p

2022 Volume 5090

Abstract: Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. ... ...

Abstract	Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. Current approaches are typically for single gene candidate peptides and often employ non-human cells. We devised an approach where multiple gene products could be produced from a single population of cells. We identified gene specific Synergistic Activation Mediators (SAM) from the CRISPR/Cas9 system for targeted overexpression of coagulation factors II, VII, IX, X, and fibrinogen. The components of the CRISPR-SAM system were expressed in Human Embryonic Kidney Cells (HEK293), and single (singleplex) or multi-gene (multiplex) upregulation was assessed by quantitative RT-PCR (qRT-PCR) and protein expression by ELISA analysis. Factor II, VII, IX, and X singleplex and multiplex activation resulted in 120–4700-fold and 60–680-fold increases in gene expression, respectively. Fibrinogen sub-unit gene activation resulted in a 1700–92,000-fold increases and 80–5500-fold increases in singleplex or multiplex approaches, respectively. ELISA analysis showed a concomitant upregulation of candidate gene products. Our findings demonstrate the capability of CRISPR/Cas9 SAMs for single or multi-agent production in human cells and represent an engineering advance that augments current recombinant peptide production techniques.
Keywords	CRISPR ; recombinant protein ; coagulation ; multiplexing ; fibrinogen ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	572
Language	English
Publishing date	2022-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics

Mark J. Osborn / Akshay Bhardwaj / Samuel P. Bingea / Friederike Knipping / Colby J. Feser / Christopher J. Lees / Daniel P. Collins / Clifford J. Steer / Bruce R. Blazar / Jakub Tolar

Bioengineering, Vol 8, Iss 2, p

2021 Volume 23

Abstract: Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially ... ...

Abstract	Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.
Keywords	CRISPR/Cas9 ; SARS-Co-V2 ; lateral flow assay ; Technology ; T ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2021-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Murine CAR19 Tregs suppress acute graft-versus-host disease and maintain graft-versus-tumor responses

Sara Bolivar-Wagers / Michael L. Loschi / Sujeong Jin / Govindarajan Thangavelu / Jemma H. Larson / Cameron S. McDonald-Hyman / Ethan G. Aguilar / Asim Saha / Brent H. Koehn / Mehrdad Hefazi / Mark J. Osborn / Michael C. Jensen / John E. Wagner / Christopher A. Pennell / Bruce R. Blazar

JCI Insight, Vol 7, Iss

2022 Volume 17

Abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg ... ...

Abstract	Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg doses are often required, necessitating substantial ex vivo or in vivo expansion that may diminish suppressor function. To enhance in vivo suppressor function, murine Tregs were transduced to express an anti–human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated, hCD19-transgenic recipients for allo-HSCT. Compared with recipients receiving control transduced Tregs, those receiving hCAR19 Tregs had a marked decrease in acute GVHD lethality. Recipient hCD19 B cells and murine hCD19 TBL12-luciferase (TBL12luc) lymphoma cells were both cleared by allogeneic hCAR19 Tregs, which was indicative of graft-versus-tumor (GVT) maintenance and potentiation. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19– murine TBL12luc cells in vitro in a perforin-dependent, granzyme B–independent manner. Importantly, cyclophosphamide-treated, hCD19-transgenic mice given hCAR19 cytotoxic T lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity that has been associated with proinflammatory cytokine release; in contrast, hCAR19 Treg suppressor function enabled avoidance of this severe complication. In conclusion, hCAR19 Tregs are a potentially novel and effective strategy to suppress GVHD without loss of GVT responses.
Keywords	Immunology ; Transplantation ; Medicine ; R
Language	English
Publishing date	2022-09-01T00:00:00Z
Publisher	American Society for Clinical investigation
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Correction of Fanconi Anemia Mutations Using Digital Genome Engineering

Christopher J. Sipe / Mitchell G. Kluesner / Samuel P. Bingea / Walker S. Lahr / Aneesha A. Andrew / Minjing Wang / Anthony P. DeFeo / Timothy L. Hinkel / Kanut Laoharawee / John E. Wagner / Margaret L. MacMillan / Gregory M. Vercellotti / Jakub Tolar / Mark J. Osborn / R. Scott McIvor / Beau R. Webber / Branden S. Moriarity

International Journal of Molecular Sciences, Vol 23, Iss 8416, p

2022 Volume 8416

Abstract: Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and ... ...

Abstract	Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.
Keywords	Fanconi anemia (FA) ; gene therapy ; base editing ; adenine base editing (ABE) ; cytosine base editing (CBE) ; CRISPR-Cas9 ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	610
Language	English
Publishing date	2022-07-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells

Karolina Skvarova Kramarzova / Mark J. Osborn / Beau R. Webber / Anthony P. DeFeo / Amber N. McElroy / Chong Jai Kim / Jakub Tolar

International Journal of Molecular Sciences, Vol 18, Iss 6, p

2017 Volume 1269

Abstract: Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such ... ...

Abstract	Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 (FANCD1) primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was employed to target and correct a FANCD1 gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase). FANCD1 function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.
Keywords	gene editing ; CRISPR/Cas9 ; Fanconi anemia ; fibroblasts ; Fanconi anemia D1 ; poly adenosine diphosphate-ribose polymerase inhibitors ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2017-06-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression

Mark J. Osborn / Christopher J. Lees / Amber N. McElroy / Sarah C. Merkel / Cindy R. Eide / Wendy Mathews / Colby J. Feser / Madison Tschann / Ron T. McElmury / Beau R. Webber / Chong Jai Kim / Bruce R. Blazar / Jakub Tolar

International Journal of Molecular Sciences, Vol 19, Iss 4, p

2018 Volume 946

Abstract: Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To ... ...

Abstract	Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.
Keywords	CRISPR/Cas9 ; recessive dystrophic epidermolysis bullosa ; transcriptional activation ; homology directed repair ; adeno-associated virus ; ubiquitous chromatin opening element ; T-cells ; cord blood ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2018-03-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Author Correction

Beau R. Webber / Cara-lin Lonetree / Mitchell G. Kluesner / Matthew J. Johnson / Emily J. Pomeroy / Miechaleen D. Diers / Walker S. Lahr / Garrett M. Draper / Nicholas J. Slipek / Branden A. Smeester / Klaus N. Lovendahl / Amber N. McElroy / Wendy R. Gordon / Mark J. Osborn / Branden S. Moriarity

Nature Communications, Vol 10, Iss 1, Pp 1-

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

2019 Volume 1

Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper. ...

Abstract	An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Keywords	Science ; Q
Language	English
Publishing date	2019-12-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

Beau R. Webber / Cara-lin Lonetree / Mitchell G. Kluesner / Matthew J. Johnson / Emily J. Pomeroy / Miechaleen D. Diers / Walker S. Lahr / Garrett M. Draper / Nicholas J. Slipek / Branden S. Smeester / Klaus N. Lovendahl / Amber N. McElroy / Wendy R. Gordon / Mark J. Osborn / Branden S. Moriarity

Nature Communications, Vol 10, Iss 1, Pp 1-

2019 Volume 10

Abstract: Multiplexed genome engineering with Cas9 can increase efficiency but also the risk of unintended alterations. Here the authors demonstrate the use of multiplexed base editors on primary T cells with reduced translocation frequency. ...

Abstract	Multiplexed genome engineering with Cas9 can increase efficiency but also the risk of unintended alterations. Here the authors demonstrate the use of multiplexed base editors on primary T cells with reduced translocation frequency.
Keywords	Science ; Q
Language	English
Publishing date	2019-11-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Author Correction

Nature Communications, Vol 10, Iss 1, Pp 1-

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

2019 Volume 1

Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper. ...

Abstract	An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Keywords	Science ; Q
Language	English
Publishing date	2019-12-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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