Hereditary transthryretin amyloidosis is a rare disease caused by mutations in the gene encoding the protein transthyretin (TTR), causing it to misfold into amyloid plaques, leading to debilitating symptoms. These amyloid plaques can deposit in various tissues including nerves of the peripheral nervous system where it can affect sensation in limbs and disfunction of blood pressure, heart rate and digestion, among others. In 2018, the FDA approved the first RNA interference drug Patisiran (Trade name: Onpattro), which targets hereditary TTR amyloidosis. Patisiran relies on lipid nanoparticles (LNPs) to deliver short interfering RNA (siRNA) that interrupts production of the mutant protein. Although LNPs have solved the delivery challenge for small RNAs, the effects of siRNA are transient. Gene editing with CRISPR/Cas9 however, can remove the mutant gene from the genome of a cell to potentially provide long lasting or permanent knockdown. There are however, unique pharmacokinetic challenges to delivering gene editing components. Researchers from Intellia Therapeutics describe their approach to overcoming these challenges in their 2018 paper in Cell Reports. They achieved over 97% reduction of serum levels of TTR that lasted for at least 12 months with a single dose in rodent models.
Overcoming challenges delivering CRISPR-based therapies
CRISPR gene editing requires both the Cas9 endonuclease and a single guide RNA (sgRNA) to associate and get transported into the nucleus to find and cut target genes. Encoding Cas9 into an mRNA allows both the mRNA and the sgRNA to be encapsulated in the same particle. This overcomes the pharmacokinetic challenge of delivering both components to the same cell at the same time. Once in the cell however, the mRNA needs to be translated to protein before it can join with the sgRNA, during which time some of the sgRNA may degrade.
To overcome this challenge, Intellia have screened numerous modifications to the sgRNA designed to resist degradation. They tested these in vitro to identify modifications that maximized editing activity. The most effective modifications were adopted for further study in vivo where one set of modifications was selected as optimal.
According to Intellia, to be clinically relevant, a CRISPR-based therapeutic needs to meet 4 basic requirements: 1) Transient expression of Cas9 to mitigate the risk of off-target edits, 2) efficient delivery of CRISRP components, 3) ability to re-dose if needed, and 4) Scalability of formulation. While viral delivery of CRISPR components is popular in research, there are concerns about complexity of production, ability to re-dose and persistent Cas9 expression and possibly genome integration of Cas9. LNPs address these concerns, particularly with mRNA as the payload, persistent expression of Cas9 is not a risk. In this paper, Intellia report a half-life of mRNA and sgRNA in plasma and liver between 2 and 2.5h. With 97% knockdown that persisted for at least 12 months, they have also demonstrated very efficacious and potent editing. Previous reports of non-viral delivery of CRISPR components have resulted in 3.5% to 35% of liver cells being edited. Here however, Intellia report over 70% of liver cells have edited. Additionally, their LNP formulation was effective not only in mice but also in rats, which are of greater clinical significance.
In terms of scalability of formulations, LNPs are much less laborious to produce than viral vectors. Additionally, nucleic acid LNPs are very modular, in that different nucleic acid therapeutic agents targeting different diseases are chemically very similar to one another, allowing equipment, LNP materials, and methodologies used for development and manufacturing to be used across different indications. Intellia have used the NanoAssemblr platform to develop the LNP system presented in this paper. The platform is highly scalable allowing optimization of formulation parameters at bench scale and carry-over of critical parameters to larger scales of production including GMP-ready systems already in place.
In all, this paper addresses numerous challenges facing the development and clinical application of CRISPR gene editing. These advances are promising for the safe and effective application of gene editing in treating genetic ailments at their molecular root cause. Many rare diseases such as hereditary ATTR amyloidosis have a genetic origin and they stand to benefit from these approaches, particularly because LNP delivery systems are modular, allowing chemically similar sgRNAs to be easily substituted to target different disease-causing genes.
The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.