Chronic lymphocytic leukemia (CLL) is the most common human leukemia and the majority of patients show a deletion in the region 13q that encodes for mir15-a and mir16-1 that are normally responsible for the targeting of the antiapoptotic B cell lymphoma 2 protein (BCL2) which is consequently overexpressed in CLL and determines this pathology. In the proposed project, we will evaluate the delivery and the uptake efficiency of mir15-a and miR-16-1 through new modified ferritin-based nanoparticles. Compared with traditional systems, the humanized Archaeal ferritin (HumAfFt) offers several advantages and may be an ideal tool for an efficient and targeted delivery of nucleic acids. This chimeric ferritin is endowed with the recognition motifs typical of Human H homopolymer and the unique oligomerization properties of Archaeoglobus fulgidus ferritin. Based on an friendly protocol of purification, it is produced in large amounts, is thermostable and can be readily modified to efficiently encapsulate small nucleic acids into its cavity. Two different approaches will be used for mir-15a and 16-1 loading: i) physical entrapment by Mg2+ induced protein polymerization around nucleic acids and ii) chemical coupling of the thiol terminated miRNA with a cysteine residue located inside the ferritin cavity. Mir15-a and mir16-1 loaded HumAFt will be then tested for uptake efficiency on MEG01 cells, a very simple and powerful model displaying the classic CLL deletion 13q to study the expression of the target genes and the consequent apoptotic induction. We observed that HumAFt deliver efficiently and under physiological conditions fluorescent probes to HeLa cells through the CD71 receptor, overexpressed in many tumoral cell lines. The key deliverable of our project is a new ferritin-based RNAs nanocarrier, as a good alternative in the delivery of nucleic acids into cancer cell of human leukemia overcoming the significant experimental deficiency of traditional transfection agents.
The major challenge of therapeutics designed to engage RNA interference pathways is their intracellular delivery to specific tissue that express the target gene. The proposed methodology, based on modified ferritin-based nanoparticles, is intended to be more versatile, cheaper and safer in comparison with current therapeutic approaches based mostly on viral transfection. Moreover, using a receptor-driven uptake, the ferritin-based nanocarriers are more stable in the extracellular environment and have a higher transfection efficiency than liposomes, thus representing a unique nanotechnological tool for cell-targeted delivery of oligonucleotides for cancer treatment. Human ferritin-based nanoparticles have been extensively studied for anticancer applications and may represent a good alternative to viral vectors or organic nanoparticles for targeted delivery of fast internalization through the transferrin receptor (TfR1) which has long been known to be overexpressed in malignant cells ii) exceptionally high stability, required for therapeutic delivery formulation, iii) ease and low cost production of recombinant proteins in E. coli cells, iv) possibility of genetic manipulation in order to confer alternative receptor recognition specificities, diverse intracellular targeting capabilities or modified internal cavity properties. For the first time, we intended to explore a humanized archaeoglobus ferritin (HumAfFt) for targeted delivery of small nucleic acids. This chimeric proteins possesses a great advantage compared to other ferritin-based nanoparticles due to the mild and reversible conditions linked to their assembly-disassembly equilibrium whose control is a prerequisite in order to achieve encapsulation of the cargo within the internal cavity. Due to the potential versatility of HumAfFt, multiple delivering strategies can be investigated by chemical modification with linkers bearing different functionalities to perform both covalent or noncovalent entrapment of miRNA molecules. In addition, our delivery system can be tested on chronic lymphocytic leukemia cell lines providing a simple and efficient read out of the potentiality of the protein-based nanocarrier. TfR1, the major gate of iron uptake, is indeed overexpressed in malignant proliferating cells, since the cell cycle enzyme ribonucleotide reductase has a strong requirement for iron. Although the normal hematopoietic cells also need iron for proliferation, it is known that most of the hematopoietic stem cells are in a quiescent state with putatively lower iron requirements. This would allow for a presumably selective effect of a miRNA-HumAfFt targeting TfR1 towards malignant cells. On the other hand, the second part of our project addresses the understanding of the molecular mechanism of the internalization pathway through a clathrin-mediated mechanism which is still not completely understood and it is demanding to further optimize the HumAfFt-based nanocarrier to expand the employment in therapeutic applications.