Base editing delivery innovation achieves proof of concept for lipid-lowering gene therapy and eye disease treatment

CRISPR-Cas technology is currently the simplest and most efficient gene editing system. As a major scientific discovery, since Harvard Medical School, the Broad Institute and the University of California San Francisco applied the system to mammalian cells in 2013 to achieve specific editing of complex genomes, CRISPR technology has developed rapidly and been widely applied, quickly entering the field of human disease treatment. Representative companies include Editas Medicine, CRISPR Therapeutics and Intellia Therapeutics.

As the application boundaries of gene editing technology continue to expand and its derivative technologies develop, the technology has moved from the initial field of genetic diseases into complex diseases such as cardiovascular and neurological diseases, as well as cancer. The CRISPR gene editing therapy for thalassemia is currently progressing rapidly and is expected to become a benchmark therapeutic product in the global clinical translation of gene editing.

As early as 2015, a research team led by Chinese scientist Huang Junjiu began exploring the clinical treatment feasibility of this technology for beta-thalassemia. The research results were selected by Nature as one of the year's top ten scientific figures globally (1), opening unlimited room for imagination in using new gene editing technologies to treat genetic diseases.

The delivery system is an important factor affecting the efficacy and safety of gene therapies. Especially for in vivo gene editing therapies, the base editing system needs to be delivered into the body while ensuring its efficiency. Adeno-associated virus (AAV) is the mainstream delivery method. However, the size of gene drugs packaged by AAV must be less than 4.8kb, while the 5.4kb base editing system ABE cannot be delivered into the body through a single AAV.

Based on innovation and exploration of the key issue of delivery systems in gene therapy, the research team of Huang Junjiu of Sun Yat-sen University recently published a research report titled "Development of Highly Efficient Dual-AAV Split Adenosine Base Editor for In Vivo Gene Therapy" in Small Methods (IF: 12) (2,3).

This study achieved a more efficient dual-AAV delivery than before, and carried out effective validation of gene targets related to liver and eye diseases in animal models, establishing an internationally leading AAV combination strategy for base editing therapy and providing a new method for precision gene therapy. This is another technological achievement with potential clinical translation value from the team's sustained investment in gene editing-related research.

There are currently two strategies that may solve the delivery problem of base editing systems. One is to use an RNA trans-splicing system, but the splicing system is very inefficient, at only 1%. The other strategy uses an intein-mediated protein splicing system, which is more efficient than the RNA splicing system.

By screening inteins from different species and matching different ABE split methods, the researchers finally screened out the two combination schemes with the highest editing efficiency, ABE-Rma573 and ABE-Rma674 (Figure 1). The researchers respectively confirmed in mouse liver and eyes that using dual-AAV delivery of this editing system can achieve highly efficient in vivo base editing (Figure 2).

Figure 1. Screening system for the construction of ABE dual-AAV vectors. Figure 2. Dual-AAV-mediated delivery of Split-ABE into mice achieves highly efficient base editing in target cells of the liver and retina.

After determining the efficient delivery strategy, the researchers first verified the effect in mouse liver based on this system. Through dual-AAV delivery of the base editing system, the researchers successfully induced A-G base editing of the PCSK9 gene in mouse liver, with an efficiency of up to 6%. Six weeks after treatment with split-ABE-Rma573, the key indicator serum PCSK9 protein decreased by nearly 50%, and serum very low-density lipoprotein and low-density lipoprotein (VLDL/LDL) decreased by nearly 25%, with no liver damage observed. PCSK9 is the most widely used therapeutic target in the cardiovascular drug development field, with enormous clinical application value, and the development of this class of new drugs is expected to benefit hundreds of millions of people worldwide.

In late 2019, Novartis spent nearly USD 10 billion to acquire The Medicines Company (4), which had been developing a small RNA nucleic acid drug targeting PCSK9. The success of this drug is expected to realize, for the first time, the vision of providing long-acting nucleic acid drugs to disease populations.

On the other hand, PCSK9 drugs based on gene base editing technology seem to better reflect the effect of one-time administration and lifelong cure. In July this year, Verve Therapeutics announced its R&D data in non-human primates at the International Society for Stem Cell Research conference: using base editing technology, it successfully turned off PCSK9 expression in the livers of cynomolgus monkeys, significantly reducing blood LDL cholesterol and triglyceride levels, with no off-target effects observed. This is also an exciting milestone in the development of gene lipid-lowering drugs (5).

Another major indication for gene editing therapy is ophthalmic diseases, such as inherited blinding retinal diseases and chronic degenerative diseases. Among them, the gene drug Luxturna developed by Spark Therapeutics is the first ophthalmic gene therapy drug approved by the FDA (6). The gene editing therapy for Leber congenital amaurosis developed by Editas Medicine, co-founded by pioneering Chinese-American gene editing scientist Zhang Feng, has also entered early clinical trials. The Huang Junjiu research team also conducted validation in ophthalmology, using this system to successfully implement A-G base editing of the rod cell characteristic gene NR2E3 related to Goldmann-Favre syndrome and the angiogenesis factor VEGFA related to age-related macular degeneration, with an editing efficiency of up to 25% in the mouse retina.

The Huang Junjiu research team has long focused on academic innovation and clinical translation in the gene editing and stem cell fields. Since achieving gene editing and base repair of thalassemia, the genetic disease with the widest affected population worldwide, in 2015, the team has also carried out scientific exploration of gene editing technology applications in the liver and ophthalmology fields. Delivery and editing efficiency are the core parts of gene editing therapy. This research result shows that this highly efficient dual-AAV base editing delivery system and its proof of concept in animal models provide more advantageous treatment options for precision gene therapy of human genetic and non-genetic diseases.

References: 1. https://www.nature.com/news/365-days-nature-s-10-1.19018 2. https://onlinelibrary.wiley.com/doi/10.1002/smtd.202000309 3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5726555/ 4. https://www.forbes.com/sites/brucelee/2019/11/26/why-novartis-is-buying-the-medicines-company-for-97-billion/ 5. https://www.businesswire.com/news/home/20200627005005/en/Verve-Therapeutics-Preclinical-Data-Presented-at-ISSCR-Annual-Meeting 6. https://www.fda.gov/news-events/press-announcements/fda-approves-novel-gene-therapy-treat-patients-rare-form-inherited-vision-loss