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Toolgen's AAV delivery could move CRISPR/Cas9 beyond genetic disease


By Shannon Ellis
Staff Writer

SHANGHAI – In the midst of a busy news week for CRISPR/Cas9 – a powerful tool that gives cut-and-paste accuracy to gene editing by using adapted immune bacteria – Toolgen Inc. has made advances of its own. The Seoul, South Korea-based company shared encouraging results in Nature Communications in a study evaluating Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 orthologue characterized to date, for efficient genome editing in vivo.

While much of the attention has been focused on the patent battle for who has the rights to use of CRISPR (clustered, regularly interspaced short palindromic repeats)/Cas9 in eukaryotic cells, with the Broad Institute of Massachusetts Institute of Technology winning an important U.S. Patent Office decision last week, small firms like Toolgen are forging ahead and experimenting with different ways to expand the use of that highly effective technology as a potential therapeutic. (See BioWorld Today, Feb. 15, 2017, Feb. 16, 2017, and Feb. 17, 2017.)

Toolgen got its start in 1999 developing genetic tools such as the CRISPR/Cas9 precursors ZFNs (zinc-finger nucleases) and TALENs (transcription activator-like effector nucleases). It was founded by Jin-soo Kim, now the director of Genome Engineering Center, Institute of Basic Sciences in Korea.

Several years ago, Toolgen's research director, Seokjoong Kim, approached the translational aspect of CRISPR/Cas9 and decided that "the most reasonable way to develop therapeutic applications fast is to combine a therapeutically proven technology and CRISPR." He determined that using a single adeno-associated virus (AAV), which had been used successfully to deliver genes into the eye and liver, was the best way forward.

But size quickly became an issue. Typically, AAV can only deliver small DNA (between 4.5 kb and 4.8 kb); the most robust and preferred Cas9 system, SpCas9, was too large. After a period of trial and error, it was decided CjCas9, though slightly less robust than SpCas9, would work well with AAV.

In the Toolgen study, CjCas9 was injected into the eye of a mouse using AAV. "It was active in vitro and in vivo in [the] mouse achieving 30 percent to 60 percent gene efficiency in the retina," Kim told BioWorld Today.

The Broad Institute spin-off, Editas Medicine Inc., is also looking at AAV delivering CRISPR/Cas9 in the eye, but for Leber congenital amaurosis, a genetic eye disorder that occurs in three births per 100,000 worldwide.

Kim said Toolgen is looking at AAV as a proven delivery technique to combine with CRISPR for support in in vivo genome editing in a specific organ. Then the aim will be to find a good target disease to show proof of principle and that the approach is safe and provides therapeutic effect.


But Toolgen is taking it up a notch. The company is looking to use CRISPR/Cas9 in the eye to reduce a vascularization phenotype, with potential in much larger ocular indications that are not genetic such as age-related macular degeneration (AMD) and diabetic retinopathy (DR), the leading causes of adult blindness.

"Our paper characterized a small Cas9 system that can fit into AAV and showed that AAV-mediated delivery of this small Cas9 can actually support quite efficient gene editing in vivo, especially in the eye. We then showed, by potentially targeting a couple of different genes related to the modulation of the vascularization phenotype, that CRISPR in vivo gene editing in the eye can support or modulate the vascularization process, which is the underlying cause of AMD and DR," explained Kim.

While that has potential in terms of opening up the CRISPR field to new areas or inquiry such as other forms of rare diseases, it presents possible regulatory challenges, particularly in Korea where no other biotechs are working on AAV. Toolgen is hoping to partner with AAV companies or experts in the U.S., where the regulators have had greater exposure to that type of delivery method.

In addition, Kim said, "using tools to change genes to cure non-genetic disease is an interesting aspect of expanding the scope of gene editing therapy. We hope to be able to push this into IND, if we feel safe and confident after large animal study. In terms of regulatory perspective and societal perspective, . . . using CRISPR for non-genetic disease is a new aspect. . . . We might need to think about it a little more deeply."

If successful, the benefits could be substantial. According to the paper's authors, "because the CjCas9 target site in the mouse Hif1a gene is perfectly conserved in the human HIF1A gene, the AAV presented in this study or its variants could be used for the treatment of human patients in the future. We expect that CjCas9 can be directed to other traditionally 'undruggable' genes or noncoding sequences to broaden the range of therapeutic targets, making the entire human genome potentially druggable."

But in the meantime, Toolgen is also hedging its regulatory bets and pursuing genetic diseases as well. The AAV delivery method for CjCas9 looks set to have applications in genetic diseases, especially in the liver, and the firm is pursuing various programs such as in hemophilia A and hemophilia B. With animal studies underway, it is planning an IND filing by the end of the year in one of those indications, Kim said.

In terms of IP, Toolgen is seeking patents in 10 countries, including China, and has already been successful in Korea and Australia. The U.S. patents it is seeking have not been granted and may have been delayed until the USPTO decided on the patent interference case that MIT's Broad Institute won against Berkeley University. Kim said he is hopeful that Toolgen's provisional patent application will benefit from that ruling. (See BioWorld Today, Sept. 20, 2016.)

"If the same interpretation holds up, and the examiner finds our invention is separate from the Berkeley invention, then our claim could be allowed," he said. "This is what we are hoping for and waiting for, but there is no guarantee."