Significant efforts have been dedicated to the synthesis and validation of the I-GENE NT according to the specifications coming from mathematical modeling. Specifically, we synthesized spherical gold nanoparticles (AuNPs) and gold nanorods (AuNRs) via seed formation and growth methodologies accompanied by ligand exchange. AuNPs and AuNRs were produced with different surface features (positive and negative net charges). We discovered that the nanotransducer surface charge strongly influences cell internalization, delivery to the nucleus, and aggregation under physiological conditions. The AuNPs and AuNRs were further modified to terminate with a Nitrilotriacetic acid (NTA) group on their surface, enabling binding to recombinant Cas proteins via His-tag affinity. Laboratory protocols for protein coupling were developed to standardize the process, paving the way for future commercial exploitation (e.g. the I-Geneer Kit, which includes a product datasheet and user manual).
The different versions of the I-GENE NT were tested for targeted applications. AuNPs with a positive net charge and NTA-terminating groups were validated as non-viral vectors for delivering the Cas9:gRNA ribonucleoprotein. Our data demonstrated that AuNP-Cas9:gRNA complexes have high stability, and biocompatibility. They show ability to spontaneously enter cells without any transfection tools, to localize in the nuclei within 1-2 hours post-administration and to perform gene editing, without causing harmful off-target effects. AuNPs with negative net charge and NTA-terminating groups showed excellent biological properties but did not achieve nuclear localization. Therefore, they were repurposed for another gene therapy application targeting cytoplasmic RNA. Specifically, AuNP-Cas13, when administered to SARS-CoV-2 infected Huh-7, showed ability to spontaneously cross cell membranes without any transfection tools, to co-localize with viral particles (autophagosomes, endosomes), and to effectively abolish SARS-CoV-2 infection when targeting RNA-dependent RNA polymerase (RdRP) and Nucleocapsid (N) protein.
Next, we focused on the light-switchability of genome editing. For this application, AuNRs with a positive net charge and NTA-terminating groups were found to be optimal for binding the dCas9:gRNA ribonucleoprotein. This strategy involves using a dimer of NTs, with one binding a sequence upstream and another binding downstream of the target site. When two plasmonic AuNRs are brought into proximity, their coupling increases, and the dimer's behaviour prevails. Under "laser on" conditions, a dramatic temperature jump (>100°C for nanoseconds or microseconds) occurs in the nanoscopic gap (less than 10 nm) between the NTs. This local heat is expected to break the double-strand DNA with millisecond precision. For radiation experiments, a dedicated laser workstation was installed, comprising an optical/epifluorescence microscope, a tunable laser, a thermal camera for bulk temperature detection, a Lab-on-chip device (LoC) for cell irradiation in suspension, optical fibres to couple the laser to the LoC, and a microfluidic workstation for cell injection and flow control. This workstation was used to irradiate zebrafish larvae. To assess the gene editing efficiency of the I-GENE NT in zebrafish, the tyrosinase gene was chosen as the target. Using the "I-GENEMatcher" software (
https://i-gene.d4science.org/group/i-genepublic/i-gene-tool(si apre in una nuova finestra)) we identified the optimal pair of gRNAs. AuNR-dCas9:gRNAs were injected into zebrafish zygotes, which then underwent the irradiation process. Data analysis revealed significant indel mutations induced by the I-GENE NT under radiation, confirming the ability of the I-GENE NT to facilitate light-switchable genome editing.
