VISUALIZATION OF FACTOR VIII WITH FLOW-CYTOMETRY AS A TOOL FOR NOVEL GENE THERAPY APPROACH IN HEMOPHILIA A

Haemophilia A is a genetic X-linked disorder, characterized by coagulation Factor VIII (FVIII) deficiency and leading to pathological bleedings. The disease occurs in a rate of 1 in 5000 males’ births. The treatment is the administration of plasma-derived or recombinant Factor VIII which is expensive and leads to the development of inhibitory antibodies in around 40% of patients affected by the severe form of the disease. The disease becomes for these patients life-threatening. New approaches to treat Haemophilia include Gene Therapy (GT). Cells corrected through genetic modifications are used to produce in Haemophilia A patients FVIII protein in a sustained manner, as long-term treatment for this disorder. The cells of choice should be persistent and equipped with the machinery for large protein assembly and secretion. So far, target cells for Haemophilia gene correction are mostly liver cells, although they are highly immunogenic and exposed to immune-mediated destruction after GT. Based on literature evidences, Bone Marrow Transplantation can correct Haemophilia A in mice, providing evidence that Hematopoietic Stem Cells (HSC) or their progeny are able to produce FVIII. We chose the approach of correcting HSC with lentiviral vectors carrying the FVIII gene cassette. Whereas classically FVIII protein is visualized on adherent cells through immunohistochemistry staining, Flow-Cytometry (FC) literature publications are very scarce. FC analysis is an attractive method for analysing hematopoietic cells, and in general, a versatile method for protein visualization. However, large proteins as FVIII are difficult to be carefully analysed, and the method requires several steps of optimization. This joint project with Dr. Muhammad Elnaggar, (postdoctoral fellow) .aims to optimize a method to characterize large proteins as FVIII with a reliable FC staining protocol. To this aim we used cell lines to evaluate the expression and secretion pathways of FVIII, the intracellular requirements to fold and secrete large proteins, and the toxicities of protein accumulation, in case of GT mediated protein overexpression. To this aim, the FC experiments were performed to optimise the FC protocol for FVIII visualization, by improving blocking efficacy, antibody labelling efficacy and to ensure accuracy and validity through qPCR and FC double staining. This FC protocol proved its validity and usefulness in visualizing and studying functionally FVIII. The project successfully facilitated safe GT protocols in HSC, moreover, addressed the need in the field of biomedical research by optimizing a reliable FC protocol of staining and visualization of large proteins like FVIII

The Current Status of Cytomegalovirus (CMV) Prevalence in the MENA region: a systematic review

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus worldwide. According to the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), CMV infects people of all ages, and by the age of five, approximately one-third of children in the United States are infected. Although the infection is generally asymptomatic, it can cause severe disease in immunocompromised patients, transplant and transfusion recipients, as well as newborn neonates. The objective of this study is to systematically review published literature on CMV in the MENA region to estimate its incidence in the region and describe its epidemiological and clinical significance. The literature was searched through four scientific databases: PubMed, Scopus, Science Direct, and Web of Science. A total of 72 studies from 11 countries satisfied the inclusion criteria, covering a period from 1988–2019. The CMV IgG seroprevalence ranged from 8.7–99.2% (SD = 38.95%). CMV incidence in these countries ranged between 1.22% and 77% in transplant and transfusion recipients, with an increase in incidence with advanced age. However, the incidence rate was unclear for congenital CMV due to the variability of the reporting. This review highlights the need for more robust and well-designed studies to better estimate CMV incidence in the MENA region, standardize diagnostic criteria, and consider prophylactic and pre-emptive treatments to limit the morbidity and mortality of the disease

Potential Adverse Effects of Resveratrol: A Literature Review.

Due to its health benefits, resveratrol (RE) is one of the most researched natural polyphenols. Resveratrol's health benefits were first highlighted in the early 1990s in the French paradox study, which opened extensive research activity into this compound. Ever since, several pharmacological activities including antioxidant, anti-aging, anti-inflammatory, anti-cancerous, anti-diabetic, cardioprotective, and neuroprotective properties, were attributed to RE. However, results from the available human clinical trials were controversial concerning the protective effects of RE against diseases and their sequelae. The reason for these conflicting findings is varied but differences in the characteristics of the enrolled patients, RE doses used, and duration of RE supplementation were proposed, at least in part, as possible causes. In particular, the optimal RE dosage capable of maximizing its health benefits without raising toxicity issues remains an area of extensive research. In this context, while there is a consistent body of literature on the protective effects of RE against diseases, there are relatively few reports investigating its possible toxicity. Indeed, toxicity and adverse effects were reported following consumption of RE; therefore, extensive future studies on the long-term effects, as well as the in vivo adverse effects, of RE supplementation in humans are needed. Furthermore, data on the interactions of RE when combined with other therapies are still lacking, as well as results related to its absorption and bioavailability in the human body. In this review, we collect and summarize the available literature about RE toxicity and side effects. In this process, we analyze in vitro and in vivo studies that have addressed this stilbenoid. These studies suggest that RE still has an unexplored side. Finally, we discuss the new delivery methods that are being employed to overcome the low bioavailability of RE

VISUALIZATION OF FACTOR VIII WITH FLOW-CYTOMETRY AS A TOOL FOR NOVEL GENE THERAPY APPROACH IN HEMOPHILIA A

Haemophilia A is a genetic X-linked disorder, characterized by coagulation Factor VIII (FVIII) deficiency and leading to pathological bleedings. The disease occurs in a rate of 1 in 5000 males' births. The treatment is the administration of plasma-derived or recombinant Factor VIII which is expensive and leads to the development of inhibitory antibodies in around 40% of patients affected by the severe form of the disease. The disease becomes for these patients life-threatening. New approaches to treat Haemophilia include Gene Therapy (GT). Cells corrected through genetic modifications are used to produce in Haemophilia A patients FVIII protein in a sustained manner, as long-term treatment for this disorder. The cells of choice should be persistent and equipped with the machinery for large protein assembly and secretion. So far, target cells for Haemophilia gene correction are mostly liver cells, although they are highly immunogenic and exposed to immune-mediated destruction after GT. Based on literature evidences, Bone Marrow Transplantation can correct Haemophilia A in mice, providing evidence that Hematopoietic Stem Cells (HSC) or their progeny are able to produce FVIII. We chose the approach of correcting HSC with lentiviral vectors carrying the FVIII gene cassette. Whereas classically FVIII protein is visualized on adherent cells through immunohistochemistry staining, Flow-Cytometry (FC) literature publications are very scarce. FC analysis is an attractive method for analysing hematopoietic cells, and in general, a versatile method for protein visualization. However, large proteins as FVIII are difficult to be carefully analysed, and the method requires several steps of optimization. This joint project with Dr. Muhammad Elnaggar, .aims to optimize a method to characterize large proteins as FVIII with a reliable FC staining protocol. To this aim we used cell lines to evaluate the expression and secretion pathways of FVIII, the intracellular requirements to fold and secrete large proteins, and the toxicities of protein accumulation, in case of GT mediated protein overexpression. To this aim, the FC experiments were performed to optimise the FC protocol for FVIII visualization, by improving blocking efficacy, antibody labelling efficacy and to ensure accuracy and validity through qPCR and FC double staining. This FC protocol proved its validity and usefulness in visualizing and studying functionally FVII

CD14+/CD31+ monocytes expanded by UM171 correct hemophilia A in zebrafish upon lentiviral gene transfer of factor VIII

Emerging gene therapy clinical trials test the correction of hemophilia A (HA) by replacing factor VIII (FVIII) in autologous hematopoietic stem cells (HSCs). Although it is known that platelets, monocyte/macrophages, and mesenchymal stromal cells can secrete transgenic FVIII, a systematic examination of blood lineages as extrahepatic sources of FVIII, to our knowledge, has not yet been performed. In this study, we sought to provide a comprehensive map of native and lentivirus-based transgenic FVIII production from HSC stage to mature blood cells, through a flow cytometry analysis. In addition, we generated a model of transient HA in zebrafish based on antisense RNA, to assess the corrective potential of the FVIII-transduced HSCs. We discovered that FVIII production begins at the CD34+ progenitor stage after cytokine stimulation in culture. Among all mature white blood cells, monocytes are the largest producers of native FVIII and can maintain protein overexpression during differentiation from HSCs when transduced by a FVIII lentiviral vector. Moreover, the addition of the HSC self-renewal agonist UM171 to CD34+ cells during transduction expanded a subpopulation of CD14+/CD31+ monocytes with excellent ability to carry the FVIII transgene, allowing the correction of HA phenotype in zebrafish. Finally, the HA zebrafish model showed that f8 RNA is predominantly localized in the hematopoietic system at the larval stage, which indicates a potential contributory role of FVIII in hematopoiesis that warrants further investigation. We believe that this study may be of broad interest to hematologists and researchers striving to advance knowledge and permanent treatments for patients with HA

Tailoring cells for clinical needs : meeting report from the advanced therapy in healthcare symposium (October 28–29 2017, Doha, Qatar)

New technologies and therapies designed to facilitate development of personalized treatments are rapidly emerging in the field of biomedicine. Strikingly, the goal of personalized medicine refined the concept of therapy by developing cell-based therapies, the so-called “living drugs”. Breakthrough advancements were achieved in this regard in the fields of gene therapy, cell therapy, tissue-engineered products and advanced therapeutic techniques. The Advanced Therapies in Healthcare symposium, organized by the Clinical Research Center Department of Sidra Medicine, in Doha, Qatar (October 2017), brought together world-renowned experts from the fields of oncology, hematology, immunology, inflammation, autoimmune disorders, and stem cells to offer a comprehensive picture of the status of worldwide advanced therapies in both pre-clinical and clinical development, providing insights to the research phase, clinical data and regulatory aspects of these therapies. Highlights of the meeting are provided in this meeting report

Proceedings From the First International Workshop at Sidra Medicine: "Engineered Immune Cells in Cancer Immunotherapy (EICCI): From Discovery to Off-the-Shelf Development", 15th-16th February 2019, Doha, Qatar

The progress in the isolation and characterization of tumor antigen (TA)-specific T lymphocytes and in the genetic modification of immune cells allowed the clinical development of adoptive cell therapy (ACT). Several clinical studies highlighted the striking clinical activity of T cells engineered to express either Chimeric Antigen (CAR) or T Cell (TCR) Receptors to target molecularly defined antigens expressed on tumor cells. The breakthrough of immunotherapy is represented by the approval of CAR-T cells specific for advanced or refractory CD19+ B cell malignancies by both the Food and Drug Administration (FDA) and the European Medicinal Agency (EMA). Moreover, advances in the manufacturing and gene editing of engineered immune cells contributed to the selection of drug products with desired phenotype, refined specificity and decreased toxicity. An important step toward the optimization of CAR-T cell therapy is the development of “off-the shelf” T cell products that allow to reduce the complexity and the costs of the manufacturing and to render these drugs available for a broad number of cancer patients. The Engineered Immune Cells in Cancer Immunotherapy (EICCI) workshop hosted in Doha, Qatar, renowned experts, from both academia and industry, to present and discuss the progress on both pre-clinical and clinical development of genetically modified immune cells, including advances in the “off-the-shelf” manufacturing. These experts have addressed also organizational needs and hurdles for the clinical grade production and application of these biological drugs