CRISPR/Cas9 Edition of the <i>F9</i> Gene in Human Mesenchymal Stem Cells for Hemophilia B Therapy
Hemophilia B is a genetic disorder characterized by clotting factor IX deficiency and bleeding in joints and muscles. Current treatments involve intravenous infusion of plasma-derived products or recombinant proteins, which have limited efficacy due to the short half-life of infused proteins. Recent...
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2024-12-01
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| author | Irving Jair Lara-Navarro Luis Felipe Jave-Suárez Juan Antonio Marchal Ana Rebeca Jaloma-Cruz |
| author_facet | Irving Jair Lara-Navarro Luis Felipe Jave-Suárez Juan Antonio Marchal Ana Rebeca Jaloma-Cruz |
| author_sort | Irving Jair Lara-Navarro |
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| description | Hemophilia B is a genetic disorder characterized by clotting factor IX deficiency and bleeding in joints and muscles. Current treatments involve intravenous infusion of plasma-derived products or recombinant proteins, which have limited efficacy due to the short half-life of infused proteins. Recently, gene therapy for bleeding disorders has offered a potential solution. This study aimed to develop an in vitro gene therapy model using the CRISPR/Cas9 system to incorporate the <i>F9</i> cDNA in human mesenchymal stem cells (hMSCs) to produce clotting factor IX. RNA guide sequences targeting the promoter-exon 1 region of the <i>F9</i> gene were designed to incorporate a wild-type <i>F9</i> cDNA into the cells. Knockin was performed with the CRISPR/Cas9 system and pDONOR-CMV/cDNAF9/IRES/EGFP vector template recombination in Lenti-X HEK293 cells and MSCs. A lentiviral <i>F9</i> cDNA vector was designed as a FIX secretor model to validate the CRISPR/Cas9 system. Results showed successful gene editing and <i>F9</i> expression in both cell models, although editing efficiency was lower in hMSCs. Future investigations will focus on improving gene editing efficiency using different transfection conditions or hybrid methodologies. This study demonstrates the potential of CRISPR/Cas9-based gene therapy in hMSCs as a target for hemophilia B. Further optimizations are required to translate these findings into clinical applications. |
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| institution | Kabale University |
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| language | English |
| publishDate | 2024-12-01 |
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| spelling | doaj-art-14b2e30c3a004e6e8a15fd54b72d4cca2024-12-27T14:36:10ZengMDPI AGLife2075-17292024-12-011412164010.3390/life14121640CRISPR/Cas9 Edition of the <i>F9</i> Gene in Human Mesenchymal Stem Cells for Hemophilia B TherapyIrving Jair Lara-Navarro0Luis Felipe Jave-Suárez1Juan Antonio Marchal2Ana Rebeca Jaloma-Cruz3Doctorado en Genética Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara 44340, Jalisco, MexicoDivisión de Inmunología, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara 44340, Jalisco, MexicoDepartamento de Anatomía y Embriología Humana, Universidad de Granada, 18012 Granada, SpainDivisión de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara 44340, Jalisco, MexicoHemophilia B is a genetic disorder characterized by clotting factor IX deficiency and bleeding in joints and muscles. Current treatments involve intravenous infusion of plasma-derived products or recombinant proteins, which have limited efficacy due to the short half-life of infused proteins. Recently, gene therapy for bleeding disorders has offered a potential solution. This study aimed to develop an in vitro gene therapy model using the CRISPR/Cas9 system to incorporate the <i>F9</i> cDNA in human mesenchymal stem cells (hMSCs) to produce clotting factor IX. RNA guide sequences targeting the promoter-exon 1 region of the <i>F9</i> gene were designed to incorporate a wild-type <i>F9</i> cDNA into the cells. Knockin was performed with the CRISPR/Cas9 system and pDONOR-CMV/cDNAF9/IRES/EGFP vector template recombination in Lenti-X HEK293 cells and MSCs. A lentiviral <i>F9</i> cDNA vector was designed as a FIX secretor model to validate the CRISPR/Cas9 system. Results showed successful gene editing and <i>F9</i> expression in both cell models, although editing efficiency was lower in hMSCs. Future investigations will focus on improving gene editing efficiency using different transfection conditions or hybrid methodologies. This study demonstrates the potential of CRISPR/Cas9-based gene therapy in hMSCs as a target for hemophilia B. Further optimizations are required to translate these findings into clinical applications.https://www.mdpi.com/2075-1729/14/12/1640hemophilia Blentiviral vectorCRISPR/Cas9 systemmesenchymal stem cellsgene therapy |
| spellingShingle | Irving Jair Lara-Navarro Luis Felipe Jave-Suárez Juan Antonio Marchal Ana Rebeca Jaloma-Cruz CRISPR/Cas9 Edition of the <i>F9</i> Gene in Human Mesenchymal Stem Cells for Hemophilia B Therapy Life hemophilia B lentiviral vector CRISPR/Cas9 system mesenchymal stem cells gene therapy |
| title | CRISPR/Cas9 Edition of the <i>F9</i> Gene in Human Mesenchymal Stem Cells for Hemophilia B Therapy |
| title_full | CRISPR/Cas9 Edition of the <i>F9</i> Gene in Human Mesenchymal Stem Cells for Hemophilia B Therapy |
| title_fullStr | CRISPR/Cas9 Edition of the <i>F9</i> Gene in Human Mesenchymal Stem Cells for Hemophilia B Therapy |
| title_full_unstemmed | CRISPR/Cas9 Edition of the <i>F9</i> Gene in Human Mesenchymal Stem Cells for Hemophilia B Therapy |
| title_short | CRISPR/Cas9 Edition of the <i>F9</i> Gene in Human Mesenchymal Stem Cells for Hemophilia B Therapy |
| title_sort | crispr cas9 edition of the i f9 i gene in human mesenchymal stem cells for hemophilia b therapy |
| topic | hemophilia B lentiviral vector CRISPR/Cas9 system mesenchymal stem cells gene therapy |
| url | https://www.mdpi.com/2075-1729/14/12/1640 |
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