Assessment of Potential Side Effects Related To RAB27A Gene Therapy in Stem Cells
Özgür Doğuş Erol1,2, Mehmet Emin Şeker1,2, Şimal Şenocak1
1Department of Stem Cell Sciences, Graduate School of Health Sciences, Center for Stem Cell Research and Development (PEDİSTEM), Hacettepe University , Ankara, Turkey.
Abstract:
RAB27A plays an essential role in the regulation of exocytosis and intracellular vesicle trafficking. Loss-of-function mutations in the RAB27A gene cause dysfunctional immune cells and Griscelli Syndrome Type 2 (GS-2), whereas upregulation of RAB27A in cancer cells is associated with a worse prognosis and increased metastasis. Here, we wanted to assess the potential side effects of overexpression of RAB27A in different types of healthy stem cells as preparation for the development of gene therapy for GS-2. Bone marrow mesenchymal stem cells (BM-MSCs) were obtained from GS-2 patients and healthy donors. Healthy murine bone marrow-derived and human cord blood-derived hematopoietic stem/progenitor cells (HSPCs) were transduced with different lentiviral vectors carrying a codon-optimized RAB27A (RAB27Aco) transgene. Cells were used for in vitro functional assays and assessed using flow cytometry, Western Blot and RT-PCR. In vivo transplantation assays in mice were used to assess the effect of RAB27A on stem cell function. Engraftment was assessed using flow cytometry, sections of BM-MSC injection sites were analyzed using histological staining. Overexpression of RAB27A resulted in phenotypic changes in BM-MSCs and decreased colony-forming capacity of HSPCs. Transplantation of RAB27A + stem cells was not associated with any tumorigenesis. Despite high expression of RAB27A in HSPCs before transplantation, RAB27A levels in peripheral blood, bone marrow, and spleen cells remained low, indicating overexpression of RAB27A may have affected the long-term reconstitution potential. Development of gene therapy for GS-2 may require fine-tuning of RAB27A expression but is not likely to be complicated by RAB27A-induced tumorigenesis.
Insights
Overexpressing RAB27A in stem cells for Griscelli Syndrome Type 2 (GS-2) gene therapy caused phenotypic changes and reduced HSPC function but did not lead to tumors. Fine-tuning RAB27A expression is key for effective GS-2 gene therapy.
Area of Science:
- Cell Biology
- Genetics
- Immunology
Background:
- RAB27A is crucial for exocytosis and vesicle trafficking.
- Mutations cause Griscelli Syndrome Type 2 (GS-2); overexpression correlates with cancer metastasis.
- Gene therapy for GS-2 requires understanding RAB27A's effects on healthy stem cells.
Purpose of the Study:
- To evaluate potential side effects of RAB27A overexpression in healthy stem cells.
- To inform the development of gene therapy for GS-2.
- To assess RAB27A's impact on stem cell function and tumorigenesis.
Main Methods:
- Obtained bone marrow mesenchymal stem cells (BM-MSCs) from GS-2 patients and healthy donors.
- Transduced healthy murine and human hematopoietic stem/progenitor cells (HSPCs) with RAB27A.
- Performed in vitro functional assays, flow cytometry, Western Blot, RT-PCR, and in vivo transplantation studies.
Main Results:
- RAB27A overexpression induced phenotypic changes in BM-MSCs and reduced HSPC colony-forming capacity.
- Transplanted RAB27A-overexpressing stem cells did not cause tumorigenesis.
- Sustained high RAB27A expression in HSPCs was not maintained post-transplantation, potentially impacting reconstitution.
Conclusions:
- RAB27A overexpression in stem cells has functional consequences but does not induce tumors.
- Gene therapy for GS-2 necessitates careful control of RAB27A expression levels.
- RAB27A-induced tumorigenesis is unlikely to complicate GS-2 gene therapy development.
