Development of gene-therapy tools for Bernard-Soulier syndrome type C treatment

Zusammenfassung

Platelets are anucleate cell fragments released from megakaryocytes, their precursor cells which reside within the bone marrow. Platelets participate in a wide variety of processes like wound healing, role in cancer, immune response, among others. However, their main and bestdefined function is in the regulation of hemostasia. Once a vascular injury occurs, endothelial cells become detached and the basal membrane, and more specifically, collagen fibers, become exposed. Circulating von Willebrand factor (VWF) adhere to these fibers forming a complex which is recognized by the platelet surface receptor GPIb-V-IX. This interaction induces a rolling effect of circulating platelets over the injury zone, till arresting them. This process induces the activation, recruitment, and aggregation of surrounding platelets over the affected area, forming a platelet plug and ending the hemorrhage when interacting with the coagulation factors. GPIb-V-IX complex results from the assembly of four subunits, GPIbα, GPIbβ, GPIX and GPV. Pathogenic variants affecting their coding genes except for GP5 results in the manifestation of an inherited platelet disorder, Bernard-Soulier Syndrome (BSS). Accordingly to the affected gene we can distinguish BSS Type A1 when the variant appears in GP1BA, Type B (GP1BB) and Type C (GP9). Significantly, GP9 exhibits a higher number of pathogenic variants that lead to BSS As a result of those pathogenic variants, GPIb-V-IX receptor is unable to fully externalize on the platelet surface. Consequently, this generates non-functional platelets that are incapable of recognizing injury zones and performing subsequent following necessary steps. Patients suffering for BSS are characterized by frequent mucocutaneous bleeding episodes and severe hemorrhages. Additionally, they experience macrothrombocytopenia, this is, reduced count of giant platelets. Pathogenic variants affecting the assembly of the receptor explains the observed bleeding phenotype. These patients uniquely can take advantage of prevention, to avoid the bleeding episodes, and palliative treatments in order to diminish the hemorrhage severity. However, they still face a lifetime disease which diminish their life quality. In exceptional cases, hematopoietic stem cells (HSCs) transplantation among siblings affected and nonaffected by BSS has been successful in reversing the disease. Nevertheless, the limited number of compatible HLA donors and the risk of alloimmunization limits its frequent use as a therapeutic approach. Utilizing the revolutionary CRISPR-Cas9 technology, we developed knockouts for each subunit coding gene in two megakaryoblastic cell lines that constitutively express the GPIb-V-IX receptor. These knockouts shed light on the importance that each subunit makes for the assemblage of the entire receptor in a physiological human microenvironment and replicated most of the phenotypes described in the literature, these are, absence or impaired GPIb-V-IX externalization for BSS driver genes. Regarding GP5-KOs, we confirmed that this gene does not impede GPIb-IX receptor externalization but decreases GPIbβ presence. This fact within the receptor assembly did not hinder its functionality by binding VWF but it was slightly reduced. As research progresses, gene therapy is becoming increasingly important in the treatment of monogenic diseases. Specifically, gene therapy utilizing integrative vectors on HSCs, which are the source of all blood cellular components, is gaining significance in the field of hematology as a curative approach for addressing malignancies at their origin. In fact, numerous diseases are currently being investigated in clinical trials, employing various treatments based on self-inactivating lentiviral vectors. These trials aim to correct primary deficiencies seen in conditions such as Wiskott-Aldrich Syndrome, SCID-X1, or ADA-SCID. Moreover, by the end of 2022, the Food and Drug Administration has already granted approval for the marketing and distribution of two gene therapies based on lentiviral vector technology. These therapies target β-Thalassemia and Cerebral Adrenoleukodystrophy, marking significant advancements in the field. Regarding BSS, numerous studies have provided compelling evidence of the phenotypical reversion and restoration of platelet functionality through the generation of in vitro-produced platelets from induced pluripotent stem cells derived from patients with GP1BA and GP1BB mutations. Furthermore, the transduction of HSCs with lentiviral vectors expressing GP1BA and GP1BB, followed by their infusion into in vivo murine models lacking their respective genes (Gp1banull and Gp1bbnull), has consistently demonstrated a recovery of bleeding times and a significant alleviation of macrothrombocytopenia. These findings have inspired us to investigate the potential suitability of BSS type C as candidate for being corrected using a similar approach. The proposed therapeutic strategy involves treating BSS type C patients harboring GP9 pathogenic variants with a self-inactivating lentiviral vector that overexpresses GP9 in the patient's HSCs. These HSCs would be treated ex vivo and subsequently reinfused into the patient once they have been corrected. To achieve stable and tissue-specific expression of GPIX, the integration of our therapeutic cassette into the patient's HSCs is crucial. These HSCs are responsible for the production of abnormal megakaryocytes (MKs) and platelets in individuals with BSS. However, due to the limited availability of patients and the uniqueness of their bleeding characteristics, it may not be feasible to isolate HSCs for testing our gene therapy tools. Therefore, alternative preclinical models are necessary to evaluate the effectiveness of our gene replacement strategies before applying them on our final target cells. Additionally, we also developed another BSS type C disease model by knocking-out GP9 in induced pluripotent stem cells (iPSCs). This model added one step more of sophistication, because, while megakaryoblastic cell model served to explore receptor biology, in this case we were able to reproduce whole hematopoietic and megakaryocytic development, from HSCs to produce MKs and platelets. We could confirm that iPSCs lacking GP9 generated in vitro giant platelets similar to BSS patients’ platelets, becoming a perfect alternative to patients’ HSCs. The next crucial step involved in our research was the development of self-inactivating lentiviral vectors (LV) that express GPIX under physiological promoters, ensuring megakaryocytic-specific expression suitable for clinical application. The previously established GP9-KO disease models served as valuable preclinical tools for assessing the functionality of our LVs in restoring GPIX expression. Remarkably, GPIX-LVs successfully transduced GP9-KO cells, leading to the recovery of GPIX expression levels comparable to wild-type (WT) cells. Importantly, this expression was tissue-specific, as it was undetectable in other non-megakaryoblastic cell lines. Furthermore, the introduction of exogenous GPIX facilitated the externalization of the remaining subunits, particularly GPIbα, which serves as the functional subunit of the complex. These genetically rescued receptors regained their functional capacity, including the recognition of soluble VWF, agglutination in the presence of ristocetin and firm adhesion to coated VWF in the presence of botrocetin. Similarly, in our BSS Type C disease model based on iPSCs, the transduction not only restored GPIX expression on the membrane of MKs but also recovered its expression in platelets, resulting in a reversion to their normal size. In both cases, the expression levels were comparable to those of WT cells. Importantly, our LVs exhibited functional activity throughout the entire differentiation process, without being subject to epigenetic silencing. Once proved the functionality of our gene therapy tools, we wanted to validate its efficacy by recovering the GPIX expression, now in our future target cells, HSCs isolated from BSS Type C patients carrying different GP9 pathogenic variants. Through its isolation from non-mobilized peripheral blood, transduction, and subsequent differentiation into MKs and platelets, we validated the reversal of the BSS phenotype ex vivo. Therefore, these results confirm the promising therapeutic potential of our gene therapy approach. Lastly, we developed a novel murine model specifically designed to evaluate the effectiveness of our LVs in restoring normal physiological functions, such as bleeding time. This model holds significant relevance as the definitive preclinical study before progressing to the clinical phase. It will not only allow us to further evaluate the efficacy of our GPIX-expressing LVs, but also assess the integrative biosafety of our LVs and evaluate the presence of possible autoantibodies against the genetically rescued GPIb-V-IX complex. The previous findings from our own research, along with relevant discoveries made by other researchers in Gp1banull and Gp1bbnull murine models, fill us with a sense of optimism regarding the potential outcomes when treating BSS Gp9null mouse models in the near future. In conclusion, the significant findings presented in this research work firmly establish Bernard-Soulier Syndrome type C as an inherited disorder that holds the potential to be cured through gene therapy strategies.