Abstract Number: PB0491
Meeting: ISTH 2020 Congress
Background: Recent advances of clinical trials for gene therapy in hemophilia B (HB) through viral vectors have reached great results in the long-term transgene expression of therapeutic factor IX (FIX). However, liver toxicity as the targeted tissue and the drop off of FIX in plasma are still limiting. Innovative gene therapy through CRISPR/Cas system has been implemented on HB with promising results in experimental models. The CRISPR/Cas system applied on mesenchymal stem cells, which show high transfection capacity and differentiation potential, could be a plausible model to correct any mutation in F9 gene from mesenchymal cells of HB patients.
Aims: Modify the F9 gene on human mesenchymal cells through CRISPR/Cas9 system to produce functional human coagulation FIX.
Methods: Guide sequences (sgRNA) for flanking the promoter-exon 1 region and production of recombination template (CMV.FIX.IRES.EGFP) were designed for F9 gene edition. Recombination template, Cas9 enzyme and sgRNAs will be transfected on cells using EGFP expression like reporter. FIX production will be quantified by ELISA assay.
Results: Based on several software, guide sequences of high scores were obtained for flanking the target region of F9 gene. As initial test, HEK293 cells were transfected with the plasmid template (prrl.CMV.FIX.IRES.EGFP), to evaluate the transfection through expression of EGFP protein by fluorescence microscopy; however, a low expression was obtained, probably due to a deficient transfection efficiency. Human FIX secreted to culture medium was quantified by ELISA (36 a 64 ng/ml), and was frozen to evaluate the clotting activity. Human mesenchymal stem cells cultures (StemPro Human Adipose-Derived Stem Cells) were established, develop, and cryopreserved for future assays.
Conclusions: Bioinformatics programs facilitate the design of relevant guide sequences for gene editing with CRISPR/Cas9 system. The low expression of EGFP protein in HEK293 cells, confirms that further tests are necessary to improve the transgene expression.
To cite this abstract in AMA style:Lara Navarro IJ, Jave Suarez LF, Marchal Corrales JA, Ramírez Solís R, Jaloma Cruz AR. Gene Therapy Model by Modification of F9 Gene through CRISPR/Cas9 for Functional Factor IX Production in Mesenchymal Stem Cells: Preliminary Results [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/gene-therapy-model-by-modification-of-f9-gene-through-crispr-cas9-for-functional-factor-ix-production-in-mesenchymal-stem-cells-preliminary-results/. Accessed December 3, 2021.
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