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Final Report Summary - PLATELET DISORDERS (Generation of human experimental models of Bernard-Soulier Syndrome and Glanzmann Disease using cellular reprogramming)

Summary description of the project objectives

The main objective of the project was to deepen our knowledge of a rare platelet disorder denominated Bernard-Soulier syndrome (BSS) through the generation of human cellular models by cell reprogramming. In addition, we proposed to design a gene therapy based treatment to rescue the functional deficiencies of the megakaryocytes and platelets from these patients.

Here I detail the updated work plan for this project:

• Work package 1 (WP 1): Generation of iPSCs from BSS patients (iPSC-BSS) and healthy donors by cell reprogramming.
• Work package 2 (WP 2): Megakaryocytic differentiation of iPSCs from BSS patients and donors.
• Work package 3 (WP 3): Development of gene therapy tools and subsequence genetic rescue of iPSCs from BSS patients
• Work package 4 (WP 4): In vivo megakaryocytic differentiation of iPSCs from BSS patients.
• Work package 5 (WP 5): We have added a new objective to further analyze the new gene therapy tools generated in WP3 in BSS patients. The new objective aims for the ex vivo rescue (with lentiviral vectors expressing a wt copy of the mutated gene) and generation of BSS peripheral blood mononuclear cells (PBMCs)-derived platelets.

Description of the work performed since the beginning of the project

The work performed since the beginning of the project is mainly focused in WP1, WP2 and WP3

• Work package 1 (WP 1): Generation of iPSCs from BSS patients (iPSC-BSS) by cell reprogramming.
In collaboration with the Hematology and Oncology service in the Hospital Universitario Morales Meseguer de Murcia-Universidad of Murcia we have generated three in vitro models of BSS by reprogramming peripheral blood mononuclear cells from these patients. Each patient presents a unique mutation in the GP9 gene, and therefore they represent three different variants of this disease. These three iPSC-BSS lines have been fully characterized both molecular and functionally.
• Work package 2 (WP 2): Megakaryocytic differentiation of iPSCs from BSS patients and donors.
In order to differentiate the iPSC-BSS lines towards first, hematopoietic progenitors and second, megakaryocytes and platelets we are following two different protocols that aim to achieve the same objective, megakaryocytic/platelet differentiation from hPSCs:
- A new differentiation protocol under “serum/feeder-free” conditions that has been recently developed and published in Dr Slukvin´s lab and optimized in our lab. This protocol consists in changing medium every two days supplemented with different cocktails of hematopoietic cytokines.
- A differentiation protocol mediated by OP-9 co-culture optimized in our laboratory. This protocol is divided in two phases. In phase I, hPSCs colonies are forced to form embryoid bodies (EB) during 15 days. In phase II, EBs will be disaggregated and co-cultured on OP-9 cells where maturation of megakaryocytes and platelet formation will take place.
• Work package 3 (WP 3): Development of gene therapy tools and subsequence genetic rescue of iPSCs from BSS patients:
We have generated new therapeutic tools to genetically restore the mutations in GPIX gene. We have followed two different approaches for this genetic rescue:
- Lentiviral vectors – we have designed GPIX overexpressing lentiviral vectors and control lentiviral vectors. We have transduced iPSC-BSS cells with this vectors and checked GPIX expression.
- CRISPR/CAS9 gene edition – we have designed specific gRNA and donor sequences (ssODN) for editing the mutation in GPIX in iPSC-BSS. ssODN are used as a template during homologous recombination and therefore the restoration of the mutated gene will occur in a low percentage in iPSC-BSS lines. After optimizing these tools in 293T cells, we have recently started gene edition in iPSC-BSS models.

Description of the main results achieved so far

• Work package 1 (WP 1): Generation of iPSCs from BSS patients (iPSC-BSS) by cell reprogramming.
Characterization of iPSC-BSS lines involved different molecular and functional analysis. First, sequencing analysis of the GPIX gene confirmed the presence of the mutation in the three lines generated. In addition, Short Tandem Repeat (STR) profiling confirmed same genetic identity between iPSC-BSS lines and original blood samples. Moreover, exogenous reprogramming factors were silenced after 7-9 passages. Importantly, iPSC-BSS lines showed normal karyotype (46, XX).
Pluripotency tests included flow cytometry analysis of protein stem cell markers SSEA3, SSEA4, Tra1-60, Tra1-81 and Oct3/4, alkaline phosphatase staining (ALP), and qRT-PCR analysis of endogenous pluripotent transcription factors OCT-4, SOX-2, NANOG and REX1. To finally confirm pluripotency, iPSC-BSS cells were in vitro spontaneously differentiated into the three germ layers by embryoid body (EB) formation and teratoma formation.
• Work package 2 (WP 2): Megakaryocytic differentiation of iPSCs from BSS patients and donors.
We are currently differentiating iPSC-BSS lines towards hematopoietic lineage, using the two protocols described above. Preliminary results show that the three models generatedare able to generate hematopoietic progenitors in a similar fashion. What we need to check now is the capacity of these cells to generate megakaryocytes and platelets
• Work package 3 (WP 3): Development of gene therapy tools and subsequence genetic rescue of iPSCs from BSS patients
- Lentiviral vectors - We have generated GPIX overexpressing iPSC-BSS lines and control iPSC-BSS by transducing cells with lentiviral particles expressing EF1a-GPIX-PGK-NEO and EF1a-Empty vector-PGK-NEO. We have demonstrated that GPIX is ectopically expressed by RT-PCR as expected.
We are currently differentiating iPSC-BSS and rescued-iPSC-BSS lines towards hematopoietic lineage, using the two protocol described above. Preliminary results show that both lines (non-rescued and rescued) are able to generate hematopoietic progenitors in a similar fashion. What we need to check now is the capacity of these cells to generate megakaryocytes and platelets
- CRISPR/CAS9 gene edition – We have first optimized the efficiency of CRISPR/CAS9 vectors in 293T cells and we have recently started the edition on one of the iPSC-BSS models by nucleofecting cells with the editing vectors. We are currently performing single cell cloning in order to select cells that have restored the mutated gene.

Expected final results and their potential impact and use
We expect that the gene therapy tools developed in our lab will rescue both number and functionality of BSS platelets derived from both BSS disease models and BSS-PBMCs (WP 5).
We strongly believe that the combination of cell reprogramming and gene therapy techniques must be considered an innovative approach for the treatment of Bernard-Soulier Syndrome patients. If this process is effective, our results will serve as a starting point for the development of cellular-gene therapy for the treatment of this human thrombopathy by transplant of autologous human megakaryopoietic progenitors genetically repaired.
The generation of iPSCs-BSS and its differentiation towards human megakaryocytes and platelets are susceptible of intellectual protection. These cells can be used as in vitro and in vivo cellular models for the study of potential specific coagulation treatments. Likewise, the design of donor vectors containing the platelet glycoproteins cDNAs (GP Ib, IIb, IIa, V and IX) found mutated in thisdisease as well as a set of genes that allow cellular selection with antibiotics, that favour megakaryocytic differentiation (for instance SCL), lineage specific promoters that control aberrant GP expression in other cell lineages, etc. can also be protected and patented.

Reported by

FUNDACION PUBLICA ANDALUZA PROGRESO Y SALUD
Spain
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