The Prime Editing system, one of the representative achievements of Professor Liu Ruqian's team at Harvard University, has recently been further developed by Dr. Gao Xin and colleagues into an upgraded gene editing system.

This upgraded gene editing system can precisely insert an entire gene into the designated location of the cell's DNA. Unlike other gene editing therapies, it does not require modifications to be made one by one for each mutation.

Through this, Gao Xin and colleagues have broken through the bottleneck of the efficiency of targeted integration of large fragments of the mammalian cell genome, and have improved the efficiency and accuracy of the integration of large gene fragments in mammalian cells, providing new technical support for gene editing and gene therapy.

Advertisement

Based on this, it is expected that in the future, a single gene editing therapy can be developed to treat diseases caused by hundreds or even thousands of different gene mutations.

In detail, in the study, Gao Xin and colleagues used protein evolution techniques to modify the enzyme DNA large serine recombinase Bxb1.The article is translated into English as follows:

By integrating Bxb1 as a programmable gene insertion, the application value of it at the pathogenic site was explored, thereby proposing a pioneering editing technology called eePASSIGE (evolved and engineered prime-editing-assisted site-specific integrase gene editing).

At the same time, explorations were also conducted in cell lines with medicinal value, such as stem cells and primary human fibroblasts.

The "Prime-editing-assisted Site-specific Integrase Gene Editing" technology (PASSIGE) is a targeted gene insertion technology previously developed by the team.

A laboratory at the Massachusetts Institute of Technology in the United States used the "Programmable Addition via Site-specific Targeting Elements" technology (PASTE), which employs prime editors and large serine recombinases to achieve targeted gene insertion of large fragments.

In this study, the research team also explored the reasons why the PASSIGE technology is superior to the PASTE technology in terms of gene insertion efficiency.Through this, they confirm: Among the existing similar methods to date, the eePASSIGE method developed this time has a good gene insertion efficiency and programmable flexibility.

This allows eePASSIGE to inject new hope for the treatment of genetic diseases and is expected to play a role in the following areas:

Firstly, it is used to transform ex vivo therapies, CAR-T immune cell therapies, and the new generation of in vivo gene therapies, thereby better treating cystic fibrosis, phenylketonuria, eye diseases, etc.

In previous cell therapies, due to the presence of lentivirus, it would cause random insertion risks in the whole genome and lead to chromosomal variations with the first-generation Cas9 and other DNA double-strand cutting enzymes.

By transforming with this method, the above problems can be avoided.Secondly, it is used to realize more synthetic biology applications.

That is, artificial gene circuits designed by humans can be constructed within living biological cells, thereby regulating gene expression in response to external stress.

It is also possible to optimize the production of medicinal proteins by modifying cell strains.

Furthermore, by inserting large regulatory elements in a targeted and efficient manner, it is possible to study the conformation and folding of the three-dimensional genome, as well as the developmental issues of organisms.

Thirdly, it is used to enhance the traits and productivity of crops.For the modification of crop traits, how to achieve targeted insertion of DNA sequences is a recognized challenge, and eePASSIGE is expected to solve this problem.

Develop new treatment methods to address various types of pathogenic mutations.

It is reported that genetic mutations in the human body (such as missense mutations and nonsense mutations, etc.) can lead to the loss of gene function, thereby causing hereditary diseases.

For a pathogenic mutation that can induce a hereditary disease, it contains many types.

For example: for the PAH gene, it is currently known that about 1000 mutations can lead to phenylketonuria; for the CFTR gene, it is currently known that about 2000 mutations can lead to cystic fibrosis.Generally speaking, for any genetic disease, the pathogenic mutations in different patients are also different.

By developing a precise and targeted large-fragment gene insertion technology, the correct sequence of the disease-causing gene can be inserted at the endogenous site.

In this way, the gene product can be expressed at a normal physiological level, thereby solving the gene function loss caused by pathogenic mutations.

That is to say, only one therapy needs to be developed to treat the vast majority of diseases with various types of mutations.

Previously, traditional gene therapy mainly relied on exogenous viral vectors for expression. Due to the lack of endogenous regulatory elements, it is easy to have the problem of overexpression of gene products, which can lead to the occurrence of diseases.At the end of 2021, Gao Xin and colleagues published a paper on the PASSIGE technology in Nature Biotechnology, achieving programmable large-fragment gene technology and applied for a U.S. patent (which has now been approved).

The working principle of PASSIGE mainly involves two steps:

The first step is to introduce a DNA serine recombinase, such as the recognition sequence attB/P of Bxb1, at a specific gene site by a prime editor and a pair of pegRNAs.

The second step is that after Bxb1 recognizes the attB/P sequence, the plasmid DNA vector can be inserted into a specific site.

With the help of prime editing, the team has filled the gap of low programmability of the DNA serine recombinase Bxb1, thereby precisely inserting a 5.6-kb gene sequence at specific sites in the human genome, such as AAVS1, and achieving an efficiency of 6.8%.They also found that although Bxb1 is a protein derived from a bacteriophage in nature, even in human cells where stable cell lines already contain the attB/P sequence, the insertion efficiency of Bxb1 is still only about 10-20%.

This indicates that the DNA recombinase activity of Bxb1 is limited, which can achieve a higher efficiency of large-scale gene insertion in human cells.

Therefore, Gao Xin and his colleagues decided to use a protein evolution method called "Phage-Assisted Continuous Evolution (PACE)" developed in the laboratory to improve the insertion efficiency of the Bxb1 recombinase.

In the study, Gao Xin encountered such a problem: how do the evolved mutants improve the insertion efficiency?

Before this, scholars in this field had not resolved the complete structure of the Bxb1 protein multimer, and naturally they could not answer the above question.At this point, AI technology came to their aid. By using AlphaFold2, they successfully predicted the structure of Bxb1 and compared it with parts of the structures of other serine recombinase family members.

Meanwhile, to improve the editing efficiency of eePASSIGE in primary human fibroblasts, they tested the DNA donor and concentration under various conditions, over and over again, and finally found the best experimental conditions.

Recently, the related paper was published in Nature Biomedical Engineering (IF 26.8) with the title "Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing."

Smriti Pandey and Gao Xin are the co-first authors, and Liu Ruqian is the corresponding author[1].

Achieving the delivery of the DNA donor will be Gao Xin's next research goal. He said, "Exogenous DNA entering the cells can easily activate the defense response of T cells and other human primary cells, thereby producing cytotoxicity."Therefore, to achieve cell therapy based on eePASSIGE, it is necessary to develop and optimize the delivery methods of DNA donors.

Additionally, to achieve efficient insertion of large gene fragments in a targeted manner within animals and patients, it is necessary to develop new types of gene therapy based on eePASSIGE.

So, the next step for Gao Xin will also combine new delivery methods such as virus-like particles to address the difficulties of delivering large molecular editors.

Neither following the beaten track nor seeking attention.In addition, the successful publication of this paper could not have been achieved without the help of the mentor.

Gao Xin said: "Because David (Liu Ruqian) is an Investigator at the Howard Hughes Medical Institute (HHMI, The Howard Hughes Medical Institute), he can submit some equipment applications to HHMI every once in a while."

At that time, Gao Xin drafted a plan on behalf of the laboratory, asking the mentor to help apply for a QX ONE ddPCR instrument.

With this instrument, it is possible to process 5 96-well plates and 480 samples in one night, which not only greatly improves the efficiency of this topic but also brings help to other projects in the group that develop methods for large fragment gene insertion.

Figure | Liu Ruqian (Source: Harvard University News Website)For many years, the Liu Lab, where Gao Xin is located, has produced numerous achievements in the fields of gene editing technology development, molecular drug development, and protein engineering modification.

In response, he said: "My mentor David has brought together a group of researchers with different backgrounds to create a team that is both creative and collaborative."

"David is passionate about his work every day. I remember once when I was in a meeting with him and some collaborators, he jokingly said that weekdays or weekends are the same to him. His enthusiasm for work has also influenced us," Gao Xin continued.

In addition, the laboratory's "strict" selection of scientific research topics has also greatly benefited Gao Xin.

He said: "My personal feeling is that the mentor neither follows the beaten track nor conducts eye-catching research for some hype, which makes people feel very grounded."Usually, the team's starting point for selecting a topic is: Why is this issue a challenge within the field? What practical significance does solving it bring? What new insights can it add to academia? What breakthroughs can it bring to existing technologies?

"Sometimes, even if the ultimate goal is not achieved, the accumulated observations and results during the process may also yield a good paper," said Gao Xin.

It is also reported that Gao Xin graduated from Northwestern University in the United States with a master's degree and from the University of Massachusetts Medical School with a doctoral degree under the guidance of Professor Erik Sontheimer. In 2020, Gao Xin joined the laboratory of Professor Liu Ruqian at Harvard University as a postdoctoral fellow.

He said, "Before joining here, I was attracted by the protein evolution system, the application of single-base editing, and the lead editing technology here."

In Professor Liu Ruqian's research group, he and his colleagues have successively developed the PASSIGE technology and the current eePASSIGE method."In the future, I am looking forward to continuing to break through in technology development and application, licensing patents to some pharmaceutical companies, thereby transforming these technologies into drugs, and bringing hope to these families by curing the pain of patients with genetic diseases," Gao Xin said in conclusion.