A single mutation in a gene can lead to rare inherited diseases, such as cystic fibrosis, Tay-Sachs, and sickle-cell anemia, and are often difficult to treat and, for most, have no cure. However, with the current advances in CRISPR-Cas9 genome engineering technology, it may be possible to introduce new immunotherapies to treat these and other inherited diseases.
One such disease, congenital amegakaryocytic thrombocytopenia (CAMT), is a rare inherited recessive disorder that occurs when there is a loss-of-function mutation in the MPL gene, consequently leading to severe thrombocytopenia (low-level of blood platelets) at birth and progressing to bone marrow failure and pancytopenia (reduction in the number of red and white blood cells, as well as platelets). Hematopoietic stem cell transplantation (HSCT) is the only curative therapy, however, 30% of patients die of bleeding complications before HSCT.
The MPL gene provides instructions for making the thrombopoietin receptor protein (Mpl), which promotes the growth and division of cells. This receptor is especially important in thrombopoiesis, the formation of platelets, and plays a role in the maintenance of hematopoietic stem cells, which develop into all the cells of the blood.
Located on the cell’s plasma membrane, Mpl becomes active when the protein thrombopoietin (Tpo) binds to it, stimulating the JAK/STAT signaling pathway, which is important for controlling the production of blood cells. Disruption of the JAK/STAT pathway leads to multiple hematopoietic diseases, such as CAMT.
In an article by Cleyrat et al., the authors identified three siblings inflicted with CAMT due to a homozygous double MPL mutation at sites K39N and W272R, resulting in complete blockage of Mpl trafficking to the plasma membrane. Further research revealed that the Mpl mutant with a single W272R mutation was retained in the ER and failed to progress to the plasma membrane, suggesting that CAMT in the three siblings is due to a single mutation.
Two functional rescue strategies for the W272R Mpl mutation were investigated; overexpression of Golgi reassembly stacking protein 55 (GRASP55) and gene editing approach using CRISPR-Cas9. Incidentally, by overexpressing GRASP55, the Mpl mutant could be moved to the plasma membrane via an autophagy-based secretory pathway where it successfully bound Tpo and partially restored the JAK/STAT pathway. In addition, using CRISPR-Cas9 gene editing to insert a wild-type MPL repair template into the umbilical cord blood CD34+ stem cells, from one of the three siblings, restored the delivery of functional Mpl to the cell surface and, consequently, was able to generate in vitro megakaryocytic colony formation in the presence of Tpo.
According to the authors, “our study provides in vitro proof‐of‐principle that MPL mutations detected in CAMT patients may be corrected by modern gene engineering methods and/or by autophagy-activating drugs.”
Modifying patient-derived stem cells using CRISPR-Cas9 gene editing technology offers hope to treat and cure hereditary hematological diseases, especially rare inherited diseases, such as CAMT, where few treatment options exist. However, there are limitations to gene editing technology, such as off-target gene insertion, gene editing efficiency, and delivery methods of the gene editing technology. In this article, the authors addressed the limitations on control cells, the human cell line K562 and primary umbilical cord blood CD34+ stem cells from StemExpress, before transfection into the CAMT patients cord blood CD34+ stem cells. As noted earlier, CAMT is a rare inherited recessive disorder and cord blood samples are collected once from a patient, making the blood sample invaluable, therefore testing was performed on control cells. Most important, optimization of CRISPR-Cas9 gene editing technology using appropriate controls is essential before entering into clinical trials.
At StemExpress, we offer high-quality cord blood CD34+ hematopoietic stem cells that can be used in studies focusing on CRISPR-Cas9 gene editing technology. Let us help with your research needs. Please contact one of our sales specialists for inquiries at 530-303-3828 or 888-415-4215.
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