The overall aim of the project is the identification of microRNAs to be used as molecular targets for personalized combined therapeutic approaches for Shwachman-Diamond Syndrome (SDS), including CRISPR/Cas9 genome editing and read-through strategy. MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation. In almost all disorders, including myelodisplastic and oncologic diseases, microRNAs were found dysregulated, and often these alterations are associated with the phenotype of the disease and/or tumor onset and progression. Accordingly, the pharmacological modulation of microRNA activity appears to be a very interesting approach in the development of new types of drugs (miRNA therapeutics) and one important research area is the possible development of miRNA therapeutics in the field of rare diseases. Recently, in order to verify possible involvement of microRNAs in the SDS phenotype, we have analyzed the microRNA pattern which differentiate Lymphoblastoid Cell Lines (LCLs) cultures derived from SDS patients from LCLs from control healthy subjects. Of relevance, in consideration of the fact that cells from SDS patients are characterized by up-regulated mTOR phosphorylation, possibly associated with down-regulation of PTEN, the relative expression of PTEN targeting microRNAs was found variable among the cells from the different SDS patients. Our data suggest that miR-34a-3p and miR-744-3p might be considered as possible targets of antimiRNA molecules, for increasing PTEN expression in SDS patients. Altogether, these data support the concept the microRNAs might participate to the phenotype of lymphoblastoid cells of SDS patients. In the miRCOMBO-SDS project we will collect plasma samples and CD34+ progenitor cells obtained from SDS patients and healthy donors in order to compare the levels of microRNAs using Next Generation Sequencing (NGS), droplet digital PCR, quantitative PCR and Western Blotting. In parallel, we will study putative binding sites on 3’UTR of SBDS transcripts, in order to identify novel microRNAs involved in SBDS gene expression regulation. This part of the study could be of interest for the development of premiRNA and antimiRNA molecules (including molecules based on Peptide Nucleic Acids, PNA) used in combination with CRISPR/Cas9-based genome editing approaches or in association with read-through correctors. For the editing of SBDS gene, we will develop a CRISPR/Cas9 based system, by designing a short guide RNA (sgRNA) and a DNA donor templates, and by using the recombinant Cas9 endonuclease. Those molecules will be transfected to LCLs and CD34+ patients derived cells. The in-vitro editing will be assessed by quantification of the levels of corrected gene and mRNAs, and the increase of SBDS protein expression. Combined treatments with CRISPR/Cas9 system and/or read-through molecules, including PTC124 (Ataluren) and Amlexanox, will be employed in the presence of the best anti-miRNA therapeutic molecules transfected to SDS cells. The combination of these strategy is the long-term objective of the miRCOMBO-SDS project and could be very interesting in order to replace/increase the expression of SBDS in cells from Shwachman-Diamond Syndrome patients, allowing personalized therapeutic protocols to be assessed in this disease.
Combined approaches for Shwachman-Diamond Syndrome: microRNA targeting, gene editing and read-through correction (miRCOMBO-SDS)
Finotti A.
2020
Abstract
The overall aim of the project is the identification of microRNAs to be used as molecular targets for personalized combined therapeutic approaches for Shwachman-Diamond Syndrome (SDS), including CRISPR/Cas9 genome editing and read-through strategy. MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation. In almost all disorders, including myelodisplastic and oncologic diseases, microRNAs were found dysregulated, and often these alterations are associated with the phenotype of the disease and/or tumor onset and progression. Accordingly, the pharmacological modulation of microRNA activity appears to be a very interesting approach in the development of new types of drugs (miRNA therapeutics) and one important research area is the possible development of miRNA therapeutics in the field of rare diseases. Recently, in order to verify possible involvement of microRNAs in the SDS phenotype, we have analyzed the microRNA pattern which differentiate Lymphoblastoid Cell Lines (LCLs) cultures derived from SDS patients from LCLs from control healthy subjects. Of relevance, in consideration of the fact that cells from SDS patients are characterized by up-regulated mTOR phosphorylation, possibly associated with down-regulation of PTEN, the relative expression of PTEN targeting microRNAs was found variable among the cells from the different SDS patients. Our data suggest that miR-34a-3p and miR-744-3p might be considered as possible targets of antimiRNA molecules, for increasing PTEN expression in SDS patients. Altogether, these data support the concept the microRNAs might participate to the phenotype of lymphoblastoid cells of SDS patients. In the miRCOMBO-SDS project we will collect plasma samples and CD34+ progenitor cells obtained from SDS patients and healthy donors in order to compare the levels of microRNAs using Next Generation Sequencing (NGS), droplet digital PCR, quantitative PCR and Western Blotting. In parallel, we will study putative binding sites on 3’UTR of SBDS transcripts, in order to identify novel microRNAs involved in SBDS gene expression regulation. This part of the study could be of interest for the development of premiRNA and antimiRNA molecules (including molecules based on Peptide Nucleic Acids, PNA) used in combination with CRISPR/Cas9-based genome editing approaches or in association with read-through correctors. For the editing of SBDS gene, we will develop a CRISPR/Cas9 based system, by designing a short guide RNA (sgRNA) and a DNA donor templates, and by using the recombinant Cas9 endonuclease. Those molecules will be transfected to LCLs and CD34+ patients derived cells. The in-vitro editing will be assessed by quantification of the levels of corrected gene and mRNAs, and the increase of SBDS protein expression. Combined treatments with CRISPR/Cas9 system and/or read-through molecules, including PTC124 (Ataluren) and Amlexanox, will be employed in the presence of the best anti-miRNA therapeutic molecules transfected to SDS cells. The combination of these strategy is the long-term objective of the miRCOMBO-SDS project and could be very interesting in order to replace/increase the expression of SBDS in cells from Shwachman-Diamond Syndrome patients, allowing personalized therapeutic protocols to be assessed in this disease.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
