Three-component nanoparticles for simultaneous “hit-and-run” gene editing and transgene delivery
Dr Stanislav Indik , University of Veterinary Medicine
Directed genome editing is a technology allowing investigators to gain more insights into the function of genes. It also holds great promises for future therapies. In the last decade, great progress in the development of the genome editing tolls has been made. Owing to the simplicity of re-targeting, RNA-guided endonucleases (RGENs) including CRISPR/Cas9, have become the most popular genome editing tools available for the majority of bioengineers. However, the lack of methods for an efficient, versatile, cell-specific easy to use delivery to cells limits the scope and scalability of the technology. Current CRISPR/Cas9 delivery methods suffer from “teething troubles” The NIH Director Francis S. Collins, M.D., Ph.D. said in 2018: “There is an urgent need to develop new tools and technologies designed to enable safe and effective genome editing in humans.”
To overcome these limitations, we developed a novel Cas9/sgRNA delivery technology. It is based on the use of a lentiviral vector as a multipotent platform for intracellular simultaneous delivery of the Cas9 protein together with a template for the synthesis of guide RNA (sgRNA). Our vision is to efficiently and safely deliver the revolutionary CRISPR/Cas9 genome editing technology to cells to 1) facilitate treatment of as many genetic diseases as possible and to 2) generate models to study genetic diseases.
Technology: The Cas9 protein packaged in lentivirus-based nanoparticles together with sgRNA and optionally with a promoter-transgene expression cassette.
Safe editing of the mammalian genome requires transient exposure of the DNA to programmable nucleases. We used a novel strategy to produce lentivirus-based nanoparticles containing 1) the Cas9 protein and 2) a sgRNA and 3) optionally a transgene, for simultaneous delivery to cells. Transduction of the nanoparticles to cells (including hard-to-manipulate cells) results in a high level of target gene disruption (up to 100%). In addition, the nanoparticles can concurrently deliver a function-conferring transgene. The three-component nanoparticles may be used for the safe genome editing approaches. Additionally, the system may be practical for the development and manufacturing of advanced medicinal products such as “off-the-shelf” CAR-T cells requiring simultaneous disruption of T cell receptor (TCR) and delivery of chimeric artificial receptor (CAR) to T lymphocytes.
- The highest transient genome editing efficiency nontoxic + does not elicit the innate immunity.
- Reduced off-targeting editing in various cell types including the hard-to-manipulate cells (e.g. primary lymphocytes).
- Simultaneous endogenous gene disruption and a transgene delivery cell type-specific targeting.
- Clinically approved lentivectors compatible with CAR-T manufacturing protocols.
- Efficient and safe genome engineering.
- Simultaneous endogenous gene disruption and a transgene delivery.
The gene editing technologies have enormous market potential (e.g. genetic diseases).
CAR T therapies greatly outpace previous methods of cancer treatment and represent a massive jump forward for curative cancer care.
We filed 2 patent applications:
Filing date: 06.05.2019
Filing date: 03.09.2019
BIORXIV preprint: A novel toolkit for the efficient delivery of Cas9/sgRNAcomplexes to chromosomes in cells (doi: doi.org/10.1101/812925