Background

There are thousands of diseases caused by aberrant DNA sequences. Traditional small molecule and biologic therapies have had limited success in treating many of these diseases because they fail to address the underlying genetic causes. Newer approaches, such as RNA therapeutics and viral gene therapy, indirectly target the defective genes related to disease, but have limitations related to toxicity, limited durability, immune-response mediated reductions in the efficacy of repeat dosing, rejection, and random genome editing, which can cause undesirable off-target effects.

Gene editing has the potential to enable the next generation of therapeutics by directly correcting the underlying defective or mutant genes. Modification of genomic DNA at a specific target site by Crispr/Cas, Transcription activator like endonuclease (TALEN) and Zinc Finger Nuclease (ZFN) systems requires 1) locating the target site; 2) editing DNA strands by introducing strand breaks or other modifications; and 3) repairing or replacing of DNA strands.

Current technologies essentially consist of:

1) Gene Locating tool – Cas9/guide RNA, TALENs, and ZFN

2) Gene Editing tool – for example DNA strand breaking-nuclease protein systems that are a part of CRISPR/Cas, TALENs, or ZFNs

3) Gene Repair tools – for gene corrections usually requires a donor DNA template

First generation tools are efficient at the first two functions, but substantial improvements to the Gene Repair tools are needed for efficient gene modifications.

Our Strategy

In human therapeutics, animal health and agricultural applications, for CRISPR-mediated systems to achieve their full potential, a significant increase from their current efficiency levels is necessary. Consequently, efficiency-enhancing platform technologies like the GeneTether TNT™ Gene Editing Platform have potential utility across a wide spectrum of applications.

Our TNT platform enables the development of therapeutics that combine all three major elements into a “tethered cargo package” – guide RNA; nuclease system; and donor DNA template.

Human Therapeutics

CRISPR/Cas systems are being applied in gene therapy for the treatment of various human diseases, including monogenic diseases, infectious diseases, cancer, and more. Some CRISPR-mediated genome-editing therapies have already entered clinical trials.

There are more than 6,600 known monogenic diseases around the world, most of which are classified as rare diseases. Among those for which CRISPR/Cas-mediated gene editing therapies are in development are β-thalassaemia, sickle cell disease, hemophilia B, retinitis pigmentosa, Duchenne muscular dystrophy, and cystic fibrosis.

In addition to their development as therapeutics, CRISPR/Cas systems have been extensively used in a variety of species, as well as various human cell lines, to establish genetically modified animals and cell models of human diseases.

Non-Human Applications

Genetic modification in animals and plants provides a way to accelerate genetic selection, including for desirable sex or sex traits, improvement in nutritional value, avoidance of allergens, and reduction or impedance of diseases. In addition, genetically modified animals or plants hold considerable potential to improve human health, with applications ranging from acting as bioreactors in the production of pharmaceutical proteins to the generation of replacement tissues or organs for use in humans.

Our Technology Platform

While the CRISPR/Cas9 and related technologies are powerful tools for developing highly precise gene editing-based therapeutics, they are inherently inefficient for making gene corrections. “Efficiency” in gene editing refers to the ratio of gene edits actually made versus the maximum number of edits that could have been made during the delivery of a CRISPR/Cas payload. The efficiency of current CRISPR/Cas systems ranges from ~0.5% – 20%. This low level of efficiency could be improved by providing a donor DNA template to be located near the site of Crispr/Cas DNA strand breaks to repair the gene by an error-free process call Homology Directed Repair (HDR).

In current CRISPR/Cas systems, this happens by what is essentially random chance. If a double strand break is created and a donor DNA template is not nearby to be used in the repair process, the break will be repaired by an error prone process called Non-homologous End Joining (NHEJ) that could cause more mutations.

GeneTether has developed a proprietary method to conjugate, or “tether,” donor DNA templates to CRISPR/Can nucleases. We refer to this as our DNA Template- Nuclease Tethering, or TNT™, Platform. In in vitro experimentation combining our TNT™ Platform technology with CRISPR/Cas, the TNT™ Platform increased editing efficiency 7-fold to >50%. We believe the ability to combine the guide RNA nuclease system; and donor DNA template into a tethered “cargo package” is important to the development of effective cell and gene therapies.

Our tethering agents are compatible with a wide range of gene editing tools to improve the efficiency not only of CRISPR/Cas, but also TALENs, and ZFNs. We are also investigating the tethering agent’s role to direct NHEJ applications for cell and gene therapy.

Our tethering agents do not add substantially to the “payload” or size of the “cargo package” that needs to be delivered into the cell. We are investigating opportunities to combine our tethering agents with cell delivery vehicles including a range of viral vectors and nanoparticles.