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  1. Article: Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation.

    Vonada, Anne / Wakefield, Leslie / Martinez, Michael / Harding, Cary O / Grompe, Markus / Tiyaboonchai, Amita

    bioRxiv : the preprint server for biology

    2023  

    Abstract: Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding ... ...

    Abstract Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit. We previously described a gene therapy method to selectively expand hepatocytes deficient in cytochrome p450 reductase (Cypor) using acetaminophen (APAP). Because Cypor is required for the transformation of APAP to a hepatotoxic metabolite, Cypor deficient cells are protected from toxicity and are able to expand following APAP-induced liver injury. Here, we apply this selection system to correct a mouse model of phenylketonuria (PKU) by cell transplantation. Hepatocytes from a wildtype donor animal were edited in vitro to create Cypor deficiency and then transplanted into PKU animals. Following selection with APAP, blood phenylalanine concentrations were fully normalized and remained stable following APAP withdrawal. Cypor-deficient hepatocytes expanded from <1% to ~14% in corrected animals, and they showed no abnormalities in blood chemistries, liver histology, or drug metabolism. We conclude that APAP-mediated selection of transplanted hepatocytes is a potential therapeutic for PKU with long-term efficacy and a favorable safety profile.
    Language English
    Publishing date 2023-08-28
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.08.27.554228
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation.

    Vonada, Anne / Wakefield, Leslie / Martinez, Michael / Harding, Cary O / Grompe, Markus / Tiyaboonchai, Amita

    Hepatology (Baltimore, Md.)

    2023  Volume 79, Issue 5, Page(s) 1088–1097

    Abstract: Background and aims: Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, the low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by ... ...

    Abstract Background and aims: Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, the low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit. We previously described a gene therapy method to selectively expand hepatocytes deficient in cytochrome p450 reductase (Cypor) using acetaminophen (APAP). Because Cypor is required for the transformation of APAP to a hepatotoxic metabolite, Cypor-deficient cells are protected from toxicity and are able to expand following APAP-induced liver injury. Here, we apply this selection system to correct a mouse model of phenylketonuria by cell transplantation.
    Approach and results: Hepatocytes from a wild-type donor animal were edited in vitro to create Cypor deficiency and then transplanted into phenylketonuric animals. Following selection with APAP, blood phenylalanine concentrations were fully normalized and remained stable following APAP withdrawal. Cypor-deficient hepatocytes expanded from < 1% to ~14% in corrected animals, and they showed no abnormalities in blood chemistries, liver histology, or drug metabolism.
    Conclusions: We conclude that APAP-mediated selection of transplanted hepatocytes is a potential therapeutic for phenylketonuria with long-term efficacy and a favorable safety profile.
    MeSH term(s) Mice ; Animals ; Acetaminophen ; Hepatocytes/metabolism ; Liver/pathology ; Phenylketonurias/metabolism ; Phenylketonurias/pathology ; Disease Models, Animal ; Chemical and Drug Induced Liver Injury/pathology ; Mice, Inbred C57BL
    Chemical Substances Acetaminophen (362O9ITL9D)
    Language English
    Publishing date 2023-10-12
    Publishing country United States
    Document type Journal Article
    ZDB-ID 604603-4
    ISSN 1527-3350 ; 0270-9139
    ISSN (online) 1527-3350
    ISSN 0270-9139
    DOI 10.1097/HEP.0000000000000631
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo.

    Tiyaboonchai, Amita / Vonada, Anne / Posey, Jeffrey / Pelz, Carl / Wakefield, Leslie / Grompe, Markus

    Nature communications

    2022  Volume 13, Issue 1, Page(s) 7391

    Abstract: Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. ...

    Abstract Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications.
    MeSH term(s) Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Homologous Recombination ; RNA, Catalytic/genetics ; Transgenes
    Chemical Substances RNA, Guide, CRISPR-Cas Systems ; RNA, Catalytic
    Language English
    Publishing date 2022-11-30
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-022-35097-5
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  4. Article ; Online: In vivo tracing of the Cytokeratin 14 lineages using self-cleaving guide RNAs and CRISPR/Cas9.

    Tiyaboonchai, Amita / Wakefield, Leslie / Vonada, Anne / May, Catherine L / Dorrell, Craig / Enicks, David / Sairavi, Anusha / Kaestner, Klaus H / Grompe, Markus

    Developmental biology

    2023  Volume 504, Page(s) 120–127

    Abstract: The current gold-standard for genetic lineage tracing in transgenic mice is based on cell-type specific expression of Cre recombinase. As an alternative, we developed a cell-type specific CRISPR/spCas9 system for lineage tracing. This method relies on ... ...

    Abstract The current gold-standard for genetic lineage tracing in transgenic mice is based on cell-type specific expression of Cre recombinase. As an alternative, we developed a cell-type specific CRISPR/spCas9 system for lineage tracing. This method relies on RNA polymerase II promoter driven self-cleaving guide RNAs (scgRNA) to achieve tissue-specificity. To demonstrate proof-of-principle for this approach a transgenic mouse was generated harbouring a knock-in of a scgRNA into the Cytokeratin 14 (Krt14) locus. Krt14 expression marks the stem cells of squamous epithelium in the skin and oral mucosa. The scgRNA targets a Stop cassette preceding a fluorescent reporter in the Ai9-tdtomato mouse. Ai9-tdtomato reporter mice harbouring this allele along with a spCas9 transgene demonstrated precise marking of the Krt14 lineage. We conclude that RNA polymerase II promoter driven scgRNAs enable the use of CRISPR/spCas9 for genetic lineage tracing.
    MeSH term(s) Animals ; Mice ; CRISPR-Cas Systems/genetics ; Integrases/genetics ; Keratin-14/genetics ; Keratin-14/metabolism ; Mice, Transgenic ; Promoter Regions, Genetic/genetics ; RNA Polymerase II/genetics ; RNA Polymerase II/metabolism
    Chemical Substances Integrases (EC 2.7.7.-) ; Keratin-14 ; RNA Polymerase II (EC 2.7.7.-) ; Krt14 protein, mouse
    Language English
    Publishing date 2023-10-07
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 1114-9
    ISSN 1095-564X ; 0012-1606
    ISSN (online) 1095-564X
    ISSN 0012-1606
    DOI 10.1016/j.ydbio.2023.09.011
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.B 508: Show issues Location:
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  5. Article ; Online: Therapeutic liver repopulation by transient acetaminophen selection of gene-modified hepatocytes.

    Vonada, Anne / Tiyaboonchai, Amita / Nygaard, Sean / Posey, Jeffrey / Peters, Alexander Mack / Winn, Shelley R / Cantore, Alessio / Naldini, Luigi / Harding, Cary O / Grompe, Markus

    Science translational medicine

    2021  Volume 13, Issue 597

    Abstract: Gene therapy by integrating vectors is promising for monogenic liver diseases, especially in children where episomal vectors remain transient. However, reaching the therapeutic threshold with genome-integrating vectors is challenging. Therefore, we ... ...

    Abstract Gene therapy by integrating vectors is promising for monogenic liver diseases, especially in children where episomal vectors remain transient. However, reaching the therapeutic threshold with genome-integrating vectors is challenging. Therefore, we developed a method to expand hepatocytes bearing therapeutic transgenes. The common fever medicine acetaminophen becomes hepatotoxic via cytochrome p450 metabolism. Lentiviral vectors with transgenes linked in cis to a
    MeSH term(s) Acetaminophen ; Animals ; Genetic Therapy ; Hepatocytes ; Liver ; Mice ; Transgenes
    Chemical Substances Acetaminophen (362O9ITL9D)
    Language English
    Publishing date 2021-06-12
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2518854-9
    ISSN 1946-6242 ; 1946-6234
    ISSN (online) 1946-6242
    ISSN 1946-6234
    DOI 10.1126/scitranslmed.abg3047
    Database MEDical Literature Analysis and Retrieval System OnLINE

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