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Developing the iTOP kit: Empowering scientists with efficient intracellular delivery of biologicals in hard-to-manipulate cell types

Periodic Reporting for period 3 - ENTRANCE (Developing the iTOP kit: Empowering scientists with efficient intracellular delivery of biologicals in hard-to-manipulate cell types)

Reporting period: 2019-07-01 to 2019-10-31

NTrans Technologies BV has developed iTOP, a novel and extremely effective method to deliver bioactive molecules (i.e. DNA, RNA, active proteins, and their combinations), into mammalian cells, including the most biologically and biomedically relevant cells (e.g. human primary and stem cells), which are notoriously difficult to manipulate. The iTOP technology has been scientifically validated (published in the top scientific journal Cell in 2015) and patented (WO2015028969 and WO2017093326). The ENTRANCE project includes the necessary steps towards market entry of the iTOP research-use-only (RUO) kit.

The ENTRANCE project has allowed NTrans to market the iTOP technology as a tool for researchers to introduce a spectrum of biomolecules into primary and stem cells. iTOP technology also has important therapeutic applications, as it can be used to safely and efficiently deliver the CRISPR/Cas9 gene editing system into clinically relevant cells and repair gene mutations that are causative of disease. NTrans is collaborating with several gene-editing companies, the largest cluster of which worldwide is housed in Boston/Cambridge, Massachusetts area. These companies are essential business partners of NTrans in the development of new therapies for genetic diseases and cancer. After presenting the iTOP technology at the CRISPR meeting in Boston in February 2017 (http://crispr-congress.com/presentations-2017/) and in May 2018, NTrans has established collaborations with US-based pharmaceutical companies and other research institutions to use the iTOP technology as their delivery method of choice. These companies are developing therapeutics and CRISPR gene-editing technologies for application in genetic diseases and cancer. The next step after evaluation studies will be to set-up US collaborations for product development and marketing based on iTOP-technology licenses and/or joint development programs. Researchers have tested our technology. We are further testing the iTOP Technology on a small scale through marketing of our first product via the “Divvly” community platform. In a next phase of the company, we will try to find CRISPR-tool suppliers that would like to in-license the iTOP Technology® developed within this project for marketing purposes.
Our goal is to further support distribution and commercialization of the iTOP technology for the research community, however, we have realised that we do not have the sales and marketing team to do this on or own, and we are therefore looking for a CRISPR supplier. The knowledge developed within this H2020-project, has allowed us to develop the iTOP Technology® in other projects for therapeutic application within ophthalmology, immune-oncology and muscular dystrophies (Duchenne Muscular Dystrophy). The mission of NTrans is to translate the unique iTOP technology into new revolutionary therapies for the treatment of genetic diseases and cancer.

For more information on NTrans Technologies please visit our website: http://www.ntranstechnologies.com/
In the first 9 months of the project, we have reformulated and optimized the iTOP reagent to make it fully chemically defined, standardizing and optimizing experimental reproducibility, efficacy and compatibility with a large variety of (GMP) cell systems. We have developed a procedure for iTOP-mediated gene editing in various adherent cell lines, including HAP1 and HEK293 cells, and studied the introduction of CRISPR/Cas9 system into several “hard-to-transfect” cell types, such as hIPSCs and T-cells. In the second reporting period (Month 10 – 22), we have further optimized the iTOP procedure, and we have developed procedures for the transduction of CRISPR/Cas9 systems in different cell lines like ARPE-19, Jurkat and HeLa cells. Furthermore, we have performed comparison of iTOP to electroporation and Lipofectamine CRISPRMAX, and generated first experimental data that will be the basis of a publication. Focus of the ENTRANCE project has shifted towards the transduction of CRISPR/Cas9 systems, and NTrans is exploring avenues to get an exclusive license to develop and market a novel CRISPR/Cas9 system developed at a Dutch University. The ENTRANCE project has allowed us to establish several collaborations with pharmaceutical companies and suppliers of gene-editing tools who have interest in the iTOP technology, which will be enforced by the combination of the iTOP delivery tool and this novel CRISPR/Cas9 system.
The ENTRANCE project will allow NTrans to market the iTOP technology as a tool for researchers to introduce a spectrum of biomolecules into primary and stem cells. iTOP technology also has important therapeutic applications, as it can be used to safely and efficiently deliver the CRISPR/Cas9 gene editing system into clinically relevant cells and repair gene mutations that are causative of disease. NTrans is collaborating with several gene-editing companies, the largest cluster of which worldwide is housed in Boston/Cambridge, Massachusetts area. These companies are essential business partners of NTrans in the development of new therapies for genetic diseases and cancer. After presenting the iTOP technology at the CRISPR meeting in Boston in February 2017 (http://crispr-congress.com/presentations-2017/) and in May 2018, NTrans has established collaborations with US-based pharmaceutical companies and other research institutions to use the iTOP technology as their delivery method of choice. These companies are developing therapeutics and CRISPR gene-editing technologies for application in genetic diseases and cancer. The next step after evaluation studies will be to set-up US collaborations for product development and marketing based on iTOP-technology licenses and/or joint development programs.
Our goal is to further support distribution and commercialization of the iTOP technology for the research community, and to develop a therapeutic platform for iTOP-based delivery of bioactive molecules. The mission of NTrans is to translate the unique iTOP technology into new revolutionary therapies for the treatment of genetic diseases and cancer.

For more information on NTrans Technologies please visit our website: http://www.ntranstechnologies.com/
Schematic representation of iTOP technology