Another important result: Bang Yao Biologics Nature Chemical Biology publishes a paper to develop a precise and safe new adenine base editor -- "ABE9"

Base editing technology was first developed by David R.
Liu's team at Harvard University, and is mainly divided into two categories: cytosine base editor (CBE) and adenine base editor (ABE), which are fused by cytosine deaminase or modified adenine deaminase and Cas9 enzyme active mutant protein (nickase Cas9
), respectively.
At present, CBE and ABE have been widely used
in many species.
However, many experimental studies have shown that they still have serious random off-target phenomena and have not been completely solved
.

On October 14, 2022, Shanghai Bangyao Biotechnology Co.
, Ltd.
(hereinafter referred to as "Bangyao Biotechnology"), which focuses on gene and cell therapy, announced that it has worked with the team of Professor Li Dali and Professor Liu Mingyao of East China Normal University to develop and name a precise and safe new adenine base editor - "ABE9", which can effectively solve various off-target hidden dangers and safety problems in the clinical application of ABE, and theoretically provide a new precise targeting tool for the correction of nearly 50% of genetic pathogenic SNVs
。 The research results were officially published
in the international academic journal Nature Chemical Biology on October 13.
It can be said that this discovery is of great significance
for improving the safety of ABE tools, broadening their scope of application, and promoting their application in gene therapy and subsequent clinical translation.

Links to papers:

It is worth mentioning that ABE9, as a new generation of the most clinically applicable products, has completed core patent applications in advance, and the future patent layout will cover the global mainstream market
.
At present, BRB Biologics has a continuous and profound patent layout accumulation in the field of ABE technology, and has been authorized for base editor patents of many families worldwide
.

New adenine base editor "ABE9": precise and safe, with great potential for clinical application

The DNA base editor is highly promising
in germplasm improvement and gene therapy because it can efficiently catalyze base switching without generating DNA double-strand breaks (DSBs) and without the need for donor DNA templates.
However, the editing efficiency of the original ABE7.
10 developed by David R.
Liu's team is not satisfactory for many targets, although David Liu and other research groups have obtained more active ABE8 (ABE8e and ABE8s) through molecular evolution, but some inherent defects of ABE have not been solved
.
For example, a large number of random RNA off-target edits induced by ABE, a bystander effect due to a wide editing window causing untargeted base changes, and ABE induces cytosine base switching
in the base sequence of a particular TCN motif.

The study is the first to demonstrate that the highly active ABE8e induces more severe bystander effects and Cas9-independent DNA and RNA off-target, and significantly improves
the editing efficiency of cytosine 。 In order to solve the accuracy problem of ABE8e, this study based on the cryo-EM structure of ABE8e was rationally designed and screened, and it was found that the introduction of key mutations L145T and N108Q (ABE9) into the active pocket of TadA-8e deaminase can significantly reduce the editing window to adenine in positions 5-6 of sgRNA while retaining high editing activity (Figure 1).

By identifying the off-target R-Loop detection of Cas9 and deep transcriptome sequencing of RNA off-target, ABE9 was found to cause almost no random DNA and RNA off-target editing (Figure 2), demonstrating a high level of application safety
.
In addition, ABE9 demonstrated extremely high in vivo editing precision and activity
in mouse and rat embryos.
Among all F0 generation mice, the control group ABE8e caused a large number of A8 onlooker edits (18/19) while editing the A5 target site, and only 5.
1%
of the mice accurately edited A5 bits.
ABE9 can accurately edit A5 (14/16), of which 25% of mice have an editing efficiency of more than 80%, and the average efficiency of all F0 mice reaches 54.
3%.

Similarly, ABE9 can target the Gaa gene to accurately produce D645G mutations to induce Pompe disease in rats, and 48.
41% of F0 generation rats contain only A6-to-G mutations at the target site, while the precision editing of ABE7.
10 accounts for only 2.
76%.

In vivo experiments in rodents have once again demonstrated the high accuracy of ABE9 and highlighted its potential for accurate construction of animal models (Figure 3).

In addition, to further evaluate the potential of ABE gene therapy, the researchers constructed cell lines containing human pathogenic SNVs with adenosine to be corrected at the fifth position of sgRNA and in multiple contiguous adenosine
.
The results show that even in such an extremely bystander-based sequence, ABE9 can accurately edit the fifth position of sgRNA to produce A-to-G conversion, and the accurate correction ability is up to 342.
5 times higher than that of ABE8e (Figure 3).

Professor Li Dali, co-corresponding author of the paper and co-founder & vice president of BangYao Biologics, said: "In this study, in order to analyze the precise editing characteristics of ABE9 in massive targets without discrimination, more than 8,000 sgRNAs and target paired libraries were used for the analysis of the editing window, and it was found that ABE9 mainly edited the A5-A6 two-position bases very accurately and without sequence preference, which proved its extensive precision editing characteristics
。 In general, ABE9 narrows the editing window to 1-2nt, almost completely eliminates off-target editing of cytosine, and more importantly, reduces DNA/RNA off-target events to the background level, realizes high-precision, low-off-target base editing, and can also be fused with Cas variants to identify different PAMs in the future, further expanding the targeting range, not only bringing new base editing tools to basic research, but more importantly, it is expected to greatly improve the safety
of future clinical applications.
"

Deeply engaged in the field of gene therapy, BangYao Biologics has made continuous breakthroughs

As one of the earliest enterprises in the world to develop and apply gene editing technology, BRB Biotech has been adhering to technological innovation since its establishment, not only constantly overcoming industry barriers for multi-pipeline strategic layout, but also committed to developing internationally leading gene editing tools and obtaining core technologies
with independent intellectual property rights.
Looking back on the pioneering road of BangYao Biologics, it is undoubtedly a process
of continuous breakthrough and innovation.
At present, the team of BRB Biologics scientists has made many important and breakthrough progress in the development of gene editing tools, gene therapy for thalassemia and CAR-T technology innovation:

In March 2019, Nature Medicine published an article and found that the use of gene editing technology to target the editing of BCL11A erythroid enhancer will re-open γ globin expression and replace the defective β globin, which is expected to achieve the purpose of eradicating β-thalassemia;

In January 2020, Cell Research published a paper to prove the feasibility and effectiveness of single-base editor editing technology targeting HBG promoter to activate the expression of hemoglobin in the fetal period for the treatment of β-thalassemia;

In March 2020, Nature Medicine published a paper demonstrating that single-base editing technology can target the erythroid enhancer of BCL11A to activate fetal hemoglobin, or edit specific mutations β hemoglobin gene, which is expected to treat hereditary blood diseases including β-thalassemia by editing autologous hematopoietic stem cells;

In May 2020, Nature Cell Biology published an article reporting that the self-developed hyCBEs series of tools have higher editing activity and wider editing windows, which have greater advantages for the treatment of β hemoglobinopathies;

In June 2020, Nature Biotechnology published a new bifunctional base editor with high specificity and safety, A&C-BEmax;

In August 2022, Nature Medicine published detailed clinical data, and BRL-101 gene therapy for children with thalassaemia has been free from blood transfusion dependence for more than 2 years, and more comprehensively interpreted the efficacy and safety characteristics of BRL-101 treatment patients;

In August 2022, Nature published an article to prove the excellent clinical safety and efficacy of BRL-201 non-viral PD1 fixed-point integrated CAR-T therapy, enabling NHL patients to survive cancer-free for up to more than 2 years
.

In this regard, Professor Liu Mingyao, founder and chairman of BangYao Biologics, said, "The development of ABE9, an efficient and accurate new adenine base editor, provides a new idea
for the synergistic optimization of the single-base editing tool ABE in terms of active window and RNA off-target risk.
Since 2020, a series of breakthroughs in the base editor and its gene therapy for genetic diseases will add another sharp tool
to gene therapy.
At present, BRB Biologics has cooperated with many domestic medical institutions and has achieved excellent clinical results
in gene editing treatment β-thalassemia, non-viral PD1 fixed-point integrated CAR-T, and UCART.
In the future, we will continue to accelerate the transformation and landing of innovative drugs to benefit patients with rare genetic diseases and malignant tumors around the world, including thalassemia! "