The World's Leading Gene Editing Technology - BiopharmaDirect

CRISPR

Gene editing, a group of technologies that give scientists the ability to change an organism's DNA, is of vital importance in cell and gene therapy. CRISPR is a revolutionary gene editing technology which has become the hottest research tool in the field of biological sciences in just a few years since its introduction in 2012. However, people may still wonder if there are any other types of gene editing technologies.

Although CRISPR was first discovered in bacteria in 1987, it was not used for gene editing until 2012. In contrast, the zinc finger nuclease (ZFN) technology, the originator of gene editing technology, was developed as early as the 1990s, followed by the transcription activator-like effector nuclease (TALEN) in 2009.

However, CRISPR has quickly become the most widely used gene editing tool for basic research due to its lower threshold, faster speed, and lower cost than ZFN/TALEN. In the past year and a half, more than a dozen gene therapies have entered the clinic, more than half of which are based on CRISPR. At present, most gene therapies focus on single-gene rare diseases and tumor immunity, and wider applications are also being explored.

Figure 1 Gene-editing pipeline takes off. (Asher Mullard, 2020)

"Like any new technology, the typical way of CRISPR development is to start with rare or extremely serious diseases for which there is no cure at this stage."
——Jennifer Doudna, Nobel Prize winner

Compared with in vivo editing, in vitro editing provides a low-risk starting point, such as better and more controllable delivery, and safety advantages such as reduced immune response. There have been multiple clinical trials targeting thalassaemia and other hereditary red blood cell diseases, for example, Sangamo's ST-400, CRISPR Therapeutics and Vertex's CTX001, which will compete with Bluebird Bio's approved therapy ZYNTEGLO.

In addition, in vitro editing also provides technical support for the development of universal cell therapy. Why the early gene editing projects are mainly focused on blood diseases? One of the reasons is that the editing for correcting mutant genes can be done in vitro. By contrast, in vivo editing is a more daunting challenge of gene therapy.

Fortunately, at least 7 companies have already started their in vivo editing attempts. Sangamo plays a leading role and strives to surpass other in vivo gene therapy products already on the market, such as Spark's Luxturna and Avexis' AVXS-101. Both Luxturna and AVXS-101 are based on adeno-associated virus (AAV) for gene delivery, which is difficult to integrate into the genome. Sangamo's SB-913 is expected to be directly inserted into the cell chromosomal DNA through three vectors. Although the SB-913 clinical trial did not reach the clinical endpoint in 2019, the data showed that a patient treated with the highest dose had initial results. Therefore, Sangamo remained optimistic and is actively improving the therapy.

Figure 2 Applications of the technology in ZFN, TALEN, and CRISPR/Cas9 gene editing. (Beatrice Xuan Ho, et al. 2018)

In addition to Precision Biosciences' ARC nuclease, CRISPR, ZFN, TALEN, and their upgraded versions (such as Beam Therapeutics' CRISPR base editor) are still the three giants of gene editing technology.

The biggest advantage of CRISPR is that it does not involve protein engineering, and is simple to operate. It has the fastest design and construction speed (a few days), low cost (within a few hundred dollars), and can achieve high throughput, so ordinary laboratories can use CRISPR/ Cas9 technology to easily realizes gene editing. However, if TALEN and ZFN were chosen, scientists often have to spend a lot of money to hand over gene editing to biological companies.

"A graduate student with basic molecular biology knowledge can use CRISPR without any obstacles."
——Jennifer Doudna, Nobel Prize winner

The above advantages play a decisive role in the popularity of CRISPR. In fact, CRISPR has quickly become a powerful helper for scientific research in laboratories around the world. But as a matter of fact, CRISPR has several major limitations that cannot be ignored, including PAM sequence limitations, host immune response, off-target effects, potential carcinogenicity, and immunogenicity and drug delivery.

Unlike the complex dimer structure of ZFN and TALEN, the CRISPR system is a monomer structure that binds through base pairing, so the specificity is low. Of course, scientists have developed various strategies to increase specificity, including using enzymes from different bacteria, recombinant Cas9 enzymes, adjusting the secondary structure of the recognition sequence in the guide RNA, etc. Furthermore, one of the flaws of Cas enzyme is its large protein size. It is challenging to deliver its genes to cells through vectors such as adeno-associated viruses commonly used in gene therapy.

Unlike CRISPR and TALEN, ZFN is not derived from a bacterial system, so it has low immunogenicity and has been verified in humans and multiple biological models. Its biggest shortcoming is the long design time, but it has been shortened from a few months to two weeks. It is precisely because of the high technical threshold that Sangamo has become the leader in the field of ZFN development. The company has been deeply involved for more than 20 years and has received support from many international pharmaceutical companies such as Pfizer, Takeda, Sanofi, Novartis, and Biogen.

As the second-generation genome editing nuclease, TALEN can avoid off-target to a greater extent than other technologies. Compared with ZFN, TALEN is simpler to operate, faster to construct, and less expensive. And compared with CRISPR, the assembly plasmid is longer and more labor-intensive, which leads to higher costs.

Figure 3 Cost per reaction using CRISPR vs. TALENs. (Manisha Samy, 2017)

It can be seen that traditional CRISPR has no absolute advantages other than low cost and simple operation, let alone the limitations of the PAM sequence. To surpass ZFN and TALEN in clinical applications, it is necessary to control off-target and improve delivery efficiency in these two key directions.

Is there any other gene editing platform that breaks through the bottlenecks and limitations of traditional technologies? Let’s have a look.

1. ARCUS - a new generation of gene editing enzymes
In addition to the above-mentioned three giants, ARCUS nuclease, which is affiliated with Precision BioSciences, has emerged in clinical gene editing therapy. ARCUS, derived from the natural genome editing enzyme-Homing endonuclease, is a new generation of genome editing artifact, which can recognize up to 40 base pairs, has a small size and unparalleled sequence specificity, and can also achieve custom editing such as editing, embedding, and deleting DNA. Judging from the initial data, the security and AAV8 carrier delivery efficiency are remarkable.

In the exploration of curing hepatitis B, Precision BioSciences has made a gratifying breakthrough: the preclinical data displayed by the American Society for Genetics and Cellular Therapy (ASGCT) in 2020 shows that the optimized ARCUS can effectively target and degrade cccDNA by 75%, and is accompanied by knockdown of hepatitis B surface Antigen.

Figure 4 ARCUS by Precision BioSciences. (Precision Biosciences, Inc. Presentation as of March 12, 2020)

2. Locus Biosciences: Cas3 targeting bacteria
Unlike editing the human genome using CRISPR-Cas9 and others, Locus, led by Tencent, is using the unique properties of CRISPR-Cas3 to target and eliminate specific bacteria to deal with related challenges including broad-spectrum antibiotics and antibiotic resistance selection. In early 2019, Locus and Janssen signed a cooperation agreement for the development, production and commercialization of CRISPR-Cas3, and the research on Escherichia coli has entered the clinical stage. Antibiotic resistance has become a global public health problem. The commercial value of Locus Biosciences remains to be seen.

3. Arbor Biotechnologies: the smallest Cas enzyme to date
Similar to Beam Therapeutics, Arbor Biotechnologies, another company founded by Feng Zhang, also focuses on the development of Cas13.

Figure 5 Beam's crispr license. (Evaluate, 2018)

Arbor’s patented Cas13d protein, although belonging to the Cas13 family, is smaller than other members. Arbor was established in 2016 and obtained the Cas13d patent in 2019. Due to limited public information, its potential is difficult to judge for the time being.

Figure 6 Arbor's patented Cas13d protein. (US10392616B2)

4. Scribe Therapeutics: Focus on the development of new CRISPR molecules
After founding Caribou Biosciences and Mammoth Biosciences, Jennifer Doudna founded Scribe Therapeutics, a company specialized in developing new CRISPR molecules that specifically develop therapeutic properties. Scribe's first technology is called X-Editing (XE) molecule, which is a highly engineered CRISPR enzyme. Compared with the current CRISPR genome editing technology, it has high specificity and delivery.

5. Massachusetts General Hospital develops CRISPR that can get rid of PAM dependence and can target at any point in the entire genome A major limitation of the application of CRISPR is the reliance on the PAM sequence. In March 2020, the Kleinstiver team published a paper titled "Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants" in the journal Science. Through genetic modification, new Cas9 protein variant named SpG and SpRY does not require specific PAM and can bind and cleave DNA, target most human genomes without restriction, and has single base pair accuracy.

Figure 7 Expanded capabilities of C-to-T base editors with SpG and SpRY to generate protective genetic variants. (Russell T Walton, et al. 2020)

6. Transposon-encoded CRISPR: The upgraded version of CRISPR challenges the "off-target" effect In 2019, Science and Nature successively introduced two new types of CRISPR. Different from the classic CRISPR, these two upgraded versions of CRISPR have chosen transposons. One of the studies is the masterpiece of the "father of CRISPR"- Professor Zhang Feng.

Unlike traditional CRISPR, the upgraded version is not a simple and crude cutting of double-stranded DNA and hopes that the homologous recombination mechanism will repair the cut site. On the contrary, transposon enables genes to "jump" and integrate them into different sites in the genome, and does not involve double-strand break and homologous recombination repair, so it can effectively avoid off-target.

Zhang Feng’s team used the Tn7 transposon with CRISPR’s Cas12 enzyme. Tn7 is guided by CRISPR to trap the molecular “scissors” and insert itself into the target genome without cutting the DNA. The success rate of gene insertion is as high as 80%.

Figure 8 Model for RNA-guided DNA transposition. (Jonathan Strecker, et al. 2019)

As can be seen from the above, new generations of gene editing tools emerge endlessly. However, in addition to its own renewal and improvement, another challenge that cannot be ignored is effective drug delivery, especially in vivo editing applications.

The more widely used delivery vector for gene editing is AAV. However, a large number of studies have shown that some patients have pre-existing immunity to AAV vectors. Patients may also develop new AAV immunity with repeated administration. Gene therapy pioneer Jim Wilson once warned of the potential safety hazards caused by AAV. A typical case is that FDA halted the clinical development of Solid Biosciences' SGT-001 twice due to safety issues.

Compared with the traditional AAV9, the AAV8 currently used by Precision BioSciences can be cleared from the blood faster to avoid immune response. But it is worth pointing out that a shortcoming of all AAVs is that they tend to be transmitted to the liver, muscles and central nervous system, which limits the types of tissues that can be effectively edited.

In addition to AAV, Intellia and partner Regeneron are exploring lipid nanoparticle (LNP) delivery systems. In the past few decades, the FDA and EMA have approved more than a dozen drugs to deliver LNP, including Onpattro, which Alnylam relied on LNP to help. Carried by LNP, the intravenous Onpattro smoothly enters the liver cells where the target is located along with the blood circulation, silences related genes and removes the pathogenic proteins deposited in the body. In the long run, as editing applications become more and more complex, making multiple cuts at a time is the next goal, so carriers with greater carrying capacity will have more potential.

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