Periodic Reporting for period 3 - I-GENE (In-vivo Gene Editing by NanotransducErs)
Reporting period: 2022-05-01 to 2024-04-30
CRISPR/Cas9 and enzyme-based editors hold promise for genome surgery by erasing harmful mutations and rewriting them into beneficial ones, but they face critical barriers related to safety. Here, we propose a new concept of genome engineering based on nanotransducers (NT). Nanotransducers are tiny particles that can convert energy into a signal. Using programmable biology approaches, I-GENE technology would implement the concept of multi-input AND gates that require nanotransducer activation and the recognition of multiple specific gene loci to make the output (gene editing) true. By uniquely recognizing the desired genomic target among any potential off-targets in the 3 billion base pairs of the human genome, I-GENE technology aims to lock onto the correct target for precise and safe gene editing, thus expanding therapeutic applications. The present project aims to: i) design the nanotransducer, ii) synthesize and characterize its activity chemically, physically, and biologically, iii) test its use as a non-viral vector for genome editing, and iv) validate the light-switchability of the genome editing. This project will provide a proof-of-concept study using non-mammalian zebrafish embryos and melanoma models.
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(opens in new window)) 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.
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(opens in new window)) 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.
Currently, the global genome editing market has applications that span from cell line engineering to animal and plant genetic engineering. It reached $7.39 billion in 2023 and is predicted to grow to around $36.07 billion by 2033. In 2023, North America dominated the global market (48.0%). This growth is driven by the rapid increase in the number of pharmaceutical and biotechnology industries, increasing government funding, technological advancements, the high prevalence of infectious diseases and cancer, and the overall rise in the production of genetically modified crops. On the other hand, stringent regulatory policies and ethical issues are major factors restraining the development of new therapeutic treatments. Obviously, advancements in the safety of genome engineering would boost social excitement and investments in gene therapy as a cure for all diseases, especially for treating patients affected by severe and otherwise untreatable diseases. The I-GENE project addresses this priority and the major impacts are related to:
1. Generation of non-viral vectors for the delivery of the CRISPR/Cas machinery. Compared to the existing solutions, I-GENE technology is a transfection-free tool, it has a low cost, and it is versatile (any Cas protein with a histidine tag can be linked). Indeed, this formulation could represent a new generation of products that can replace the standard transfection tools on the market for biotechnological applications and the standard vectors for the delivery of Cas proteins for biomedical applications.
2. Validation of a paradigm of controllable medicine for inducible genome editing. The knowledge gained in the I-GENE project paves the way for the expansion of therapeutic applications. The bottlenecks are safety (there are ethical concerns related to the in vivo approaches because of the off-target activity of Cas proteins) and the high cost (€2.5 million for a single drug per patient), which makes this technology inaccessible to all. The I-GENE project has validated a technology that is safe because the drug can be administered as a pro-drug that can be activated by light, and cheap because it does not require viral vectors or cell-based therapies. The intellectual property resulting from the project has been protected by patents and will certainly change the current state of the art of genome editing tools.
1. Generation of non-viral vectors for the delivery of the CRISPR/Cas machinery. Compared to the existing solutions, I-GENE technology is a transfection-free tool, it has a low cost, and it is versatile (any Cas protein with a histidine tag can be linked). Indeed, this formulation could represent a new generation of products that can replace the standard transfection tools on the market for biotechnological applications and the standard vectors for the delivery of Cas proteins for biomedical applications.
2. Validation of a paradigm of controllable medicine for inducible genome editing. The knowledge gained in the I-GENE project paves the way for the expansion of therapeutic applications. The bottlenecks are safety (there are ethical concerns related to the in vivo approaches because of the off-target activity of Cas proteins) and the high cost (€2.5 million for a single drug per patient), which makes this technology inaccessible to all. The I-GENE project has validated a technology that is safe because the drug can be administered as a pro-drug that can be activated by light, and cheap because it does not require viral vectors or cell-based therapies. The intellectual property resulting from the project has been protected by patents and will certainly change the current state of the art of genome editing tools.