Advances in the Diagnosis and Gene Therapy of Congenital dyserythropoietic Anemia

Project Information

ADGENTHE-CDA

Grant agreement ID: 894737

DOI

10.3030/894737

Project closed

EC signature date 16 March 2020

Start date 16 March 2021

End date 15 March 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 172 932,48

EU contribution

€ 172 932,48

Coordinated by

BLOODGENETICS SL
Spain

Periodic Reporting for period 1 - ADGENTHE-CDA (Advances in the Diagnosis and Gene Therapy of Congenital dyserythropoietic Anemia)

Reporting period: 2021-03-16 to 2023-03-15

Summary of the context and overall objectives of the project

Hereditary haematological diseases (HHD) comprise a large and heterogeneous group of rare disorders for which easy-to apply tools for diagnosis, clinical management and patient stratification are currently lacking. A big number of patients remain without a final diagnosis due to the genetic complexity and high diagnostic costs. Therefore, there is an urgent need to provide a fast and accurate diagnosis of HHD for applying an earlier and adequate treatment. The emergence of next-generation sequencing technology (NGS) has revolutionized the diagnosis of genetic diseases including HHD providing a fast and accurate diagnosis. This proposal is focused in a particular HHD, the Congenital Dyserithropoietic Anemias (CDA), a bone marrow failure condition characterized by congenital anemia with ineffective erythropoiesis. The onset of CDA usually occurs in childhood or during the first years of adult age. The identification of mutated genes involved in CDA has improved the diagnostic possibilities and allowed a better classification of patients. Nevertheless, about 40% are unsolved CDAs due to the ignorance of the causative genes. BloodGenetics (BG) S.L. has previously identified new mutations and new causative genes of HHD and nowadays is commercializing NGS-panels for a number of HHD. Gene therapy is starting to be successfully applied in HHD.
This project is important for society because it will result in both the achievement of a faster and more accurate diagnosis of CDA and the establishment of molecular tools for CDA gene therapy, which overall will dramatically improve patients’ quality of life.

Benefits for society:
-Enhanced innovative capacities in developmenting and commercializimg a novel and improved Gene Panel for diagnosis of CDA.
- Enables completion of preclinical experimentation steps for gene therapy for CDA type II.
-Addressing rare diseases like CDA and establishing links with Spanish CDA Patient Association.

Objectives:
1. Establish a new genetic panel with novel genes for CDA diagnosis
2. Develop engineered cellular models for CDA type II.
3. Develop gene therapy tools for type II CDA in collaboration with UIC and CIEMAT.

Conclusions:
The project has significantly benefitted society by enhancing innovative capacities through the development and commercialization of a novel Gene Panel for CDA diagnosis. Additionally, it has paved the way for advancing gene therapy for CDA type II by enabling the completion of preclinical experimentation steps. Moreover, the project's focus on addressing rare diseases like CDA and establishing links with the Spanish CDA Patient Association underscores its commitment to improving healthcare outcomes and patient support. Overall, the project has successfully accomplished its objectives and milestones, marking a significant step forward in the field of CDA research, diagnosis and treatment.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Results:
1.Optimized genetic diagnosis panel (CDA panel 10040v17) developed and validated by BLOODGENETICS. NGS genetic studies conducted on 24 suspected CDA patients.
2. Eight LCLs generated at UIC from type II CDA patients.
3. LCLs from type II CDA patients expressed 50% less SEC23B protein than control cell line (Western blot studies).
4. PB samples from healthy donors and CDA II patients used for in vitro erythroid differentiation model. Resulted in decreased cell growth, increased bi- or multinucleated erythroid cells, and reduced enucleated cells.
5. Different sgRNAs for SEC23B gene designed and tested, highly effective for gene expression elimination.
6. Efficient gene editing determined in K562 cell line via RNP complexes formed between sgRNAs and Cas9 protein.
7.CB-CD34+ cells genetically edited, showing close to 90% reduction in SEC23B expression. Also observed reduced cell proliferation, appearance of bi- or multinucleated erythroid cells, and less hypoglycosylation of erythroid proteins.
8. Lentiviral vectors designed expressing wild-type or codon-optimized SEC23B cDNA under hPGK promoter. Evaluated in various cell lines and human hematopoietic progenitors.
9. In vitro studies conducted on functionality of produced lentiviral vectors.
10. Lentiviral vector transduction observed to correct phenotype in SEC23BKO hematopoietic progenitors and progenitors from CDA patients, confirming capability to correct main features of CDA II.

Dissemination:
Publications
1. Hernández G, …, Musri M, et al. Genes (Basel). 2021 Dec 13;12(12):1980. doi: 10.3390/genes12121980. PMID: 34946929; PMCID: PMC8702017.
2. Musri MM, et al. Int J Mol Sci. 2023 Jun 9;24(12):9935. doi: 10.3390/ijms24129935. PMID: 37373084; PMCID: PMC10298408.
Publication in preparation:
-Modelling congenital dyserythropoietic anemia type II through gene editing in hematopoietic stem and progenitor cells for lentiviral gene therapy development. Mercedes Dessy-Rodriguez, …Melina Mara Musri, et al. Manuscript in preparation-
Attendance and participation to 2 congresses (see details in report)

Exploitable results include:
1.Commercialization of a new panel for diagnosis in CDA, this result has been protected by creating a utility model based on the industrial secret. Due to our particular business model our utility model is based on industrial secrecy, not a patent, as human genes are not patentable. Indeed, we currently use for our diagnosis services the new panel designed thanks to this Marie Curie action.
2.For the protection policy of the data derived from the gene therapy studies (intellectual properties), we obtaining of an Orphan Drug Designation (ODD) and the intellectual property protection of the therapeutic developments achieved. To reach this stage additional preclinical experiments in vivo are needed.
3.Concrete agreements have been already signed for the protection of the relative contribution between the different parts. BG and the external collaborators of this project (CIEMAT and UIC) have already signed a collaboration agreement establishing the conditions of possible patent agreements.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

With the use of state-of-the-art technology such as NGS and optimized bioinformatic algorithms we have advanced in the establishment of a quick and effective diagnosis of CDA. In addition, we have set up new engineered cellular models using CRISPR/CAS9 system for CDA modelling, a highly necessary forward step to study and understand this disease. Finally, we have developed gene therapy tools applied to type II CDA in collaboration with International University of Catalunya (UIC) and Center for Energy, Environmental and Technological Research (CIEMAT).This project has improved the patient’s quality of life by shortening the waiting times between the onset of symptoms and their diagnosis. We also hope to contribute to the development of gene therapy for CDA type II that would lead to the total cure of the patient. These results have a high socio-economic impact, reducing the costs derived of a lack of diagnosis and treatment that usually lead to frequent hospital visits by patients.

ADGENTHE-CDA