Dottorato in BIOCHIMICA

Titolo della tesi: Engineered human ferritins for the selective delivery of bioactive molecules to pathological sites

In this PhD thesis we focused our attention on drug-delivery of bioactive molecules to pathological sites, especially in cancer and in rare genetic disorders. In particular, we have evaluated several aspects of a drug-delivery platform based the human ferritin (HFt) protein. HFt is a spherical protein able to bind to and being internalized through the transferrin receptor 1 (CD71 or TfR1), that could be exploited to load different bioactive molecules into its cavity and deliver them to the pathological cells that often overexpress CD71. We first focused on the innovative HFt-based construct, named The-0504. This nanoformulation effectively encapsulates the anticancer drug Genz-644282, significantly enhancing its cytotoxic properties. In this PhD thesis we extend our previous studies on The-0504 by: (a) testing DNA damage in vitro, (b) treating eight additional tumor xenograft models in vivo with The-0504; (c) performing pharmacokinetic (PK) studies in rats; and (d) evaluating The-0504 anti-tumor xenotransplant efficacy by optimizing its administration schedule based on PK considerations. Immunofluorescence demonstrated that The-0504 induces foci expressing the DNA double-strand break marker γH2AX more efficiently than free Genz-644282. Results of in vivo studies confirmed a remarkable anti-tumor activity of The-0504, resulting in tumor eradication in most murine xenograft models. After pharmacokinetic studies, we proved a long persistence of The-0504 in rat serum (half-life of about 40 h as compared to 15 h of the free drug). On this basis, we reduced The-0504 administration frequency from twice to once per week, with no appreciable loss in therapeutic efficacy in mice. Thanks to these positive results, we can affirm that The-0504 may be a good candidate for further clinical development in a tumor histotype-agnostic setting. To address the challenge of drug accumulation and penetration at the tumor site(s), herein we describe a first-in-class nanovector containing 24 copies each of two bioactive peptides (BAPs) genetically fused in frame to the 24 N-termini of a HFt construct, named THE-10. The two BAPs are specific for PD-L1, and integrin αVβ3/αVβ5 plus Neuropilin (iRGD) respectively, conferring immune checkpoint blockade and drug-internalization properties. In turn, the THE-10 backbone brings 48 BAPs contiguous for synergism, prolonged blood half-life, and release into the tumor microenvironment upon conditional cleavage of a metalloprotease-sensitive site. Predicted THE-10 multitasking activity was experimentally supported as follows. Size-exclusion chromatography and surface plasmon resonance demonstrated BAP cleavage/release and receptor binding (nanomolar KD). Live-cell/time-lapse imaging demonstrated 4-fold-increased internalization of naked therapeutic antibodies, mirrored by enhanced cytotoxicity of the corresponding Antibody-Drug Conjugate. A moderate antitumor effect was observed in vivo by treating immune checkpoint-sensitive syngeneic mouse colorectal model with THE-10 alone. Drug boosting was instead considerable on colorectal and pancreatic tumor allografts when THE-10 was co-administered with both small and large chemotherapeutic agents, outperforming the original iRGD cyclic peptide. Thus, THE-10 may enhance target therapy, chemotherapy and immunotherapy altogether, e.g. it candidates as a multitasking, all-round, antineoplastic therapy booster. For the final objective of this PhD thesis, we aimed to harness the advantageous properties of engineered HFt for the transport of nucleic acids (NAs), which are essential for NA interference (NAi). The delivery of small molecules like siRNAs and ASOs faces several challenges. We developed a functionalized nanovector, named HFt-HIS-PASE, specifically designed for delivering selected siRNAs and ASOs. HFt-HIS-PASE construct includes a tag of five histidine residues to facilitate endosomal escape and has been used to deliver either a modified siRNA (named si4) or ASO (named lncMB3 GapmeR #1) for the therapy of the Crouzon syndrome or Medulloblastoma pathology, respectively. In particular, we conjugated si4 to the external surface of HFt-HIS-PASE using an SPDP linker. In vitro experiments validated the therapeutic efficacy of HFt-HIS-PASE-si4 on target cells and demonstrated the modulation of FGFR2 signalling. In contrast, a pH disassembly/reassembly procedure was used to encapsulate the lncMB3 GapmeR #1 within the internal cavity of HFt-HIS-PASE. The in vitro experiments proved the biological activity of this complex resulted in approximately a 35% reduction in lncMB3 steady-state levels. This research provides the first in vitro evidence for a non-invasive therapeutic approach for both Crouzon syndrome and Medulloblastoma. Given its inherent versatility, this delivery system holds promise for treating a range of conditions linked to gene-related diseases, including rare genetic syndromes, neurological disorders, and cancer therapies.