August 17, 2022

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The crisis of CAR-T therapy:  T cells reproduce chromosomal abnormalities after Cas9 gene editing

The crisis of CAR-T therapy:  T cells reproduce chromosomal abnormalities after Cas9 gene editing



 

The crisis of CAR-T therapy:  T cells reproduce chromosomal abnormalities after Cas9 gene editing

Chromosomal aneuploidy (increase or decrease in the number of chromosomes) and chromosomal truncation are common consequences of cleavage of DNA double-strands during CRISPR-Cas9 gene editing. minimize its risk.


In 2017, the US FDA approved the launch of the first CAR-T cell therapy. Humans entered the era of cell therapy. CAR-T cell therapy has achieved good clinical results in blood cancers.

 

At present, several CAR-T therapies that have been marketed all use autologous T cells from cancer patients .

The advantage of autologous cell therapy is that it can function in the patient’s body for a long time without causing rejection.

There are many limitations, autologous cell therapy is time consuming, some acute leukemia patients do not have enough time to wait, and many seriously ill patients do not have enough T cells to engineer and do not have enough time to wait.

 

Therefore, many research teams and companies have begun to develop allogeneic cell therapy .

The cell sources of allogeneic cell therapy are more diverse, which can be peripheral blood , umbilical cord blood , and artificially induced pluripotent stem cells (iPSC) , etc.

This method is more Easy to mass-produce and less time-consuming, it can address many of the limitations of autologous cell therapy, so-called ” off-the -shelf ” cell therapy.

 

Allogeneic T cell therapy, represented by off-the-shelf CAR-T therapy, usually uses gene editing technology (mainly CRISPR-Cas9) to knock out T cell receptor (TCR) and other genes to avoid transplantation after transplantation Germ -versus-host disease (GvHD) .

 

Researchers from Tel Aviv University in Israel published a research paper entitled: Frequent aneuploidy in primary human T cells after CRISPR–Cas9 cleavage in Nature Biotechnology .

 

The study used single-cell RNA sequencing technology to verify the results of CRISPR-Cas9 gene editing of human primary T cells, and the results showed that the chromosomes where the target genes edited by CRISPR-Cas9 were located had frequent chromosomal aneuploidy (chromosomal number increase or decrease) and chromosomal truncation.

 

The findings of this study suggest that chromosomal aneuploidy and truncation are common consequences of CRISPR-Cas9 cleavage of DNA double-strands , and therefore, in clinical trials using CRISPR-Cas9 gene editing, especially CRISPR-Cas9 – based In cell therapy development, special care should be taken to monitor and minimize this potentially serious chromosomal aberration.

 

The crisis of CAR-T therapy:  T cells reproduce chromosomal abnormalities after Cas9 gene editing

 

 

In February 2020, Professor Carl June , the father of CAR-T, and others published a paper in the journal Science , reporting the results of the first human clinical trial of gene editing-based CAR-T therapy for cancer .

 

This study knocked out TCR and PD-1 on T cells by CRISPR-Cas9 gene editing. The alpha chain of the TCR protein (TCRα) is expressed by the TRAC gene, which is located on chromosome 14; the beta chain of the TCR protein (TCRβ) is expressed by TRBC.

Gene expression, which is located on chromosome 7; PD-1 protein is expressed by the PDCD1 gene, which is located on chromosome 2.

 

The crisis of CAR-T therapy:  T cells reproduce chromosomal abnormalities after Cas9 gene editing

The picture comes from the Science paper [2]

 

 

In this latest paper, the research team used CRISPR-Cas9 gene editing technology to knock out the genes expressing TCR and PD-1 in human primary T cells, and used the same gRNA as in the above Science paper to target the knockout TCR and PD-1.

The edited human primary T cells were then investigated using single-cell RNA sequencing.

 

The experimental results showed that a considerable proportion of primary T cells developed chromosomal abnormalities 4 days after gene editing.

Specifically, 9% of primary T cells had a deletion of chromosome 14, and 1.4% of primary T cells had a gain of chromosome 14, and the gene expressing TCRα was located on chromosome 14. 9.9% of primary T cells had chromosome 7 truncation , and the gene expressing TCRβ was located on chromosome 7.

After 11 days of editing, 0.9% of primary T cells still had chromosome 14 deletions, further confirming the long-term existence of this chromosomal variation.

 

This study shows that chromosomal aneuploidy (increase or decrease in the number of chromosomes) and chromosomal truncation are common consequences of cleavage of DNA double-strands during CRISPR-Cas9 gene editing. Check accordingly and try to minimize the risk.

 

A large number of studies have previously found and confirmed that CRISPR-Cas9 gene editing, due to the DNA double-strand break caused by Cas9, leads to severe chromosomal abnormalities such as deletion of large segments of chromosomes, chromosome fragmentation, and rearrangement in cells .

These findings remind us that we should strengthen the assessment and monitoring of the potential risks of CRISPR gene editing technology.

 

It should be pointed out that base editing (Base Editing) can perform gene editing without causing DNA double-strand breaks.

Beam therapeutics founded by the founder of base editing technology, Professor Liu Ruqian , as well as domestic Beth Bio , etc., are being approved by Base editing technology develops universal cell therapy, which theoretically can avoid chromosomal abnormalities caused by CRISPR-Cas9 gene editing and has better safety.

 

 

 

 

The crisis of CAR-T therapy:  T cells reproduce chromosomal abnormalities after Cas9 gene editing

(source:internet, reference only)


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