Periodic Reporting for period 2 - I-GENE (In-vivo Gene Editing by NanotransducErs)
Berichtszeitraum: 2020-11-01 bis 2022-04-30
CRISPR/Cas9 and enzyme-based editors hold promise for genome surgery by erasing harmful mutations while re-writing them in helpful ones, but face critical barriers related to safety. Here, we propose a new concept of genome engineering based on nanotranducers. Nanotransducers are tiny particles that can convert energy into a signal. Using approaches of programmable biology, 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. Thanks to unique recognition of the desired genomic target from any potential off-target in the 3 billion base pairs of the human genome, I-GENE technology plans to lock in the correct target for precise and safe gene editing and expanded therapeutic use. In the present project, the nanotransducer will be designed, developed and experimentally validated. Proof of concept studies for technology optimization will be performed on non-mammalian zebrafish embryos. Subsequently, the therapeutic potential will be validated in a murine model of melanoma.
The I-GENE nanotransducer (NT) has been designed to be a hybrid system that includes a DNA recognition element (hereafter, the enzyme) with the ability to guide the nanotransducer (NT) in the desired genomic location and a plasmonic gold nanoparticle (AuNP) that is highly tuned to absorb a specific optical wavelength and efficiently convert it into energy. The energy generated by the NT will be used for a thermo-inducible DNA break.
Most of the efforts of the 1st reporting period were devoted to the synthesis of the NT, its chemical, physical and biological characterisation and to simulate its plasmonic behaviour in the water environment during radiation. Prochimia Surfaces synthesised the AuNPs via its seed formation and grow methodology accompanied by ligand exchange. These AuNPs have a very narrow distribution and high stability. They have been functionalised with two kinds of distal functional group ligands for subsequent covalent linkage to the enzyme. UNIPI confirmed the ability of AuNPs to efficiently conjugate the enzyme. Furthermore, the NT is not cytotoxic and possesses the ability to localize to the correct genomic location. The NT also possess ability of spontaneous cell internalization and nuclear localization, to some degree, but these features need further improvements. Upon preparation, a long term storage of the NT at 4°C (up to several weeks) is possible. The Consortium is now working on the production and optimisation of all the NT components to develop a proprietary formulation for pushing future commercial exploitation of this product.
Many project efforts have been focused on mathematical simulations. The simulation activity of IIT has produced a clear representation of the process and has well-defined the experimental conditions allowing to obtain the parameters (radiation wavelength, power density, time) required for NT activation. We also made advances in the development of our technological solutions: the development of the laser workstation and the lab-on-chip. M Squared worked to design the laser configuration and the optical schemes for inducing NT activation in the radiated samples (cells and living organisms), according to specifications coming from simulation studies. A lab-on-chip device was designed by Lionix which follows the requirements set in the project. The configuration was conceived for handling cells in suspension (previously electroporated with the NTs) that enter the fluidic channel, are exposed to the laser radiation for milliseconds for NT activation and, consequently, induction of I-GENE genome editing.
Most of the efforts of the 1st reporting period were devoted to the synthesis of the NT, its chemical, physical and biological characterisation and to simulate its plasmonic behaviour in the water environment during radiation. Prochimia Surfaces synthesised the AuNPs via its seed formation and grow methodology accompanied by ligand exchange. These AuNPs have a very narrow distribution and high stability. They have been functionalised with two kinds of distal functional group ligands for subsequent covalent linkage to the enzyme. UNIPI confirmed the ability of AuNPs to efficiently conjugate the enzyme. Furthermore, the NT is not cytotoxic and possesses the ability to localize to the correct genomic location. The NT also possess ability of spontaneous cell internalization and nuclear localization, to some degree, but these features need further improvements. Upon preparation, a long term storage of the NT at 4°C (up to several weeks) is possible. The Consortium is now working on the production and optimisation of all the NT components to develop a proprietary formulation for pushing future commercial exploitation of this product.
Many project efforts have been focused on mathematical simulations. The simulation activity of IIT has produced a clear representation of the process and has well-defined the experimental conditions allowing to obtain the parameters (radiation wavelength, power density, time) required for NT activation. We also made advances in the development of our technological solutions: the development of the laser workstation and the lab-on-chip. M Squared worked to design the laser configuration and the optical schemes for inducing NT activation in the radiated samples (cells and living organisms), according to specifications coming from simulation studies. A lab-on-chip device was designed by Lionix which follows the requirements set in the project. The configuration was conceived for handling cells in suspension (previously electroporated with the NTs) that enter the fluidic channel, are exposed to the laser radiation for milliseconds for NT activation and, consequently, induction of I-GENE genome editing.
Currently, the global genome editing market has applications that span from cell line engineering to animal and plant genetic engineering. It is expected to reach $$6.3 billion by 2022, at a CAGR of 14.5% during (2019-2022), driven by the rapid increase in the number of pharmaceutical and biotechnology industries, increasing government funding, technological advancements, high prevalence of infectious diseases, 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 growth of this market, especially in the healthcare sector. Obviously, advancement 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. I-GENE project would address this priority. The expected results of I-GENE project is to prove that approaches of programmable biology and advances in material science can be used to develop a radically new concept of genome engineering that allow a safe implementation of gene therapy, whose technology goes behind current knowledge protected by US CRISPR/Cas9 patents.
I-Gene project intend to achieve the expected impact through four different impact pathways:
● Conceptual development, by providing a new paradigm of genome editing, characterized by safety and low cost, relative to other methods
● Technology development, by providing new vectors, devices and software for genome editing available for marketing
● Economical development, removing barriers to widespread adoption of genome editing and creating new opportunities and applications for EU industry, especially in healthcare
● Policy influence, removing/revisit ethical concern, safety issues of genome editing, which will make policy makers, public authorities and international organisations more aware of the possibility of a safe genome editing, contributing to the development of a European genome editing strategy ethically acceptable, sustainable and societally desirable.
I-Gene project intend to achieve the expected impact through four different impact pathways:
● Conceptual development, by providing a new paradigm of genome editing, characterized by safety and low cost, relative to other methods
● Technology development, by providing new vectors, devices and software for genome editing available for marketing
● Economical development, removing barriers to widespread adoption of genome editing and creating new opportunities and applications for EU industry, especially in healthcare
● Policy influence, removing/revisit ethical concern, safety issues of genome editing, which will make policy makers, public authorities and international organisations more aware of the possibility of a safe genome editing, contributing to the development of a European genome editing strategy ethically acceptable, sustainable and societally desirable.