Optimization and Risk Assessment of RAB27A-/- Gene Therapy Alternatives Using Current Molecular Techniques and Technologies

Abstract

EROL DUYAR, Ö.D. Optimization and Risk Assessment of RAB27A-/- Gene Therapy Alternatives using Current Molecular Techniques and Technologies. Hacettepe University Graduate School of Health Sciences Stem Cell Doctor of Philosophy Thesis, Ankara, 2024. Griscelli Syndrome Type 2 (GS-2) is caused by a mutation in RAB27A which plays a role in exocytosis and membrane trafficking, resulting in immune deficiency. Here, we investigated 1) the efficacy of CRISPR/Cas9 gene editing of RAB27A using GS-2 MSCs and iPSC lines; 2) the efficacy of LV vectors to transduce MSCs and express RAB27A under the control of the SFFV, PGK and UCOE promoters, and 3) potential risks related to overexpression of RAB27A in immune deficient mice. We designed CRISPR/Cas9 constructs that target mutations in RAB27A and tested the gene correction efficacy on GS-2 MSC and iPSC lines. We generated a new GS-2 iPSC line and optimized cell culture/cryopreservation. Although editing of RAB27A using CRISPR/Cas9 is possible, survival of the stem cells was low. Transduction of GS-2 MSCs with LV vectors showed the highest RAB27A expression with SFFV, followed by PGK and UCOE. RAB27A+ MSCs and HSCs were transplanted into immune deficient mice but did not cause tumor formation. However, RAB27A overexpression may affect stem cell function. In conclusion, we compared the efficiency of gene editing using CRISPR/Cas9 with gene addition using LV vectors for the development of gene therapy for GS-2. Although gene editing results in acceptable levels of repair, the technology results in low cell viability. LV gene transfer was easy and robust, but high expression of RAB27A affected stem cell function. Thus, both methods can be developed into gene therapy for GS-2, but optimization of the procedures, increasing cell viability and fine-tuning of expression levels may be necessary before these therapies are ready for clinical use. Keywords: Griscelli Syndrome Type 2, CRISPR/Cas9, lentivirus, stem cells. (*) This study was supported by grants from the Scientific and Technological Research Council of Turkish Government TÜBİTAK no: 219S675 and Hacettepe University Scientific Research Projects Coordination Unit grant no: TUK-2019-17760 and THD-2022-19940.