CRISPR-Edited T cells Evaluated in Combating Lung Cancer

By Sahana Shankar, Ph.D. Candidate

CRISPR-Cas9 has been a revolution in biological research for at least a decade now. It is routinely used in laboratory procedures to create mutants, add/delete genes in model cell lines, identify disease targets, and develop transgenic organisms. The gene-editing technology has been hailed as the holy grail of gene therapy, enabling disease cures by removing/replacing the causative aberrant genes. However, clinical applications have been scarce due to limitations in delivery methods, off-target effects, efficacy, the fitness of edited cells, and immunogenicity of CRISPR components.

CRISPR-Cas9

CRISPR (clusters of regularly interspaced short palindromic repeats) was identified in bacteria as an evolved immune response against viral attacks. It is a specialized region of DNA comprising nucleotide repeats and spacers. The bacteria stores small fragments of Viral DNA sequences from previous infections as CRISPR arrays in place of spacers.

When the virus is encountered again, the CRISPR array identifies the foreign DNA and induces efficient viral clearance. This is done by producing an RNA from the array that complementarily binds to the viral DNA facilitating cleavage by the Cas9 nuclease enzyme. This naturally occurring mechanism was adopted as a gene editing technique where the Cas9 can be guided to edit out a gene of interest using guide RNAs.

CRISPR-based gene therapies have been in trials for various genetic diseases such as Duchenne muscular dystrophy, hemophilia, β-Thalassemia, cystic fibrosis, and multiple cancers. Lung cancer is the most aggressive cancer with the highest fatality rates. Targeted immune therapy has shown positive results in treating various lung cancers due to the availability of good biomarkers.

CRISPR Usage to Treat Lung Cancer

PD-1 (Programmed Death-1) and its ligand, PD-L1 are important biomarkers of Non-Small Cell Lung Cancer (NSCLC). Increased expression of PD-1 inhibits the T cell immune response. In healthy individuals, PD-1 prevents an autoimmune response to host cells. In cancer, PD-1 activity prevents T cell response and helps tumor progression. Inhibitors of PD-1 are routinely used in therapy with good results.

A recent study, published in Nature Medicine, led by oncologist, You Lu at the West China Hospital in Chengdu, explored the possibility of disrupting the PD-1 gene in T-cells of NSCLC patients and replacing them with CRISPR-edited cells to improve therapeutic efficiency. By enrolling PD-L1+ patients in advanced stages of NSCLC who had failed multiple rounds of chemotherapy, the authors sought to test the feasibility of gene-edited T cell therapy. This is one in a series of PD-1 edited T cell therapy for lung cancer, pioneered by Dr. Lu, who was the first to conduct a human clinical trial in 2016.

T-cells extracted from peripheral blood of the patient were edited to disrupt the PD-1 gene, expanded ex vivo, and re-infused into the patient in 2 cycles of 3 infusions, 28 days apart. To confirm no off-target editing occurred, Next Generation Sequencing of the amplified PD-1 gene and whole genome sequencing was performed with an off-target mutation frequency of 0.05%. A majority of these were in the intergenic and intronic regions. The edited cells were tracked in the patient’s body to understand their T cell receptor (TCR) clone diversity.

Of the 22 patients enrolled in the clinical trial, 12 received edited T-cells (on an average of 1 billion T cells). 2 sgRNAs (single-guided RNA) were used to target PD-1 and transfected into T cells by electroporation. An average editing efficiency of 5.9% was evaluated by multiple tests such as the T7E1 cleavage surveyor assay, TA-clone sequencing, and NGS-targeted PD-1 sequencing. Edited cells had decreased PD-1 expression, stable viability, and higher populations of CD3+ and CD8+ T cells.

Median progression-free survival in 12 patients was 7.7 weeks and the median overall survival was 42.6 weeks. 90% of patients who received the edited T cells showed some mild adverse events such as lymphopenia, fatigue, leukopenia, fever, arthralgia, rash, neutropenia, etc. Clinical outcomes were characterized by patient B-01, a 55-y/o woman with metastatic lung adenocarcinoma.

After 54 weeks of T cell infusion, there was a minimal residual tumor and increased infiltrating T-cells and CD68+ macrophages with a median PD-1 editing efficiency of 12.5%, unique persistent TCR clones, and TCR diversity during 17 months of clinical follow-up. This indicates that CRISPR edited T cell gene therapy can be a useful method of intervention for lung cancer.

The authors also discussed (a) the varied response of enrolled patients which may be due to insufficient T cell expansion ex vivo, lack of antigen specificity and PD-L1-independent tumor cells, (b) low editing efficiency by electroporation- newer and better methods have been in practice which offer better efficiency. Further clinical trials with RNP (ribonucleoprotein) editing may further improve therapeutic efficacy. This study could be a proof of concept that CRISPR-Cas9 gene-edited T cell therapy to target the PD-1 gene is clinically feasible in advanced lung cancer patients with limited off-target effects.

Editor: Rajaneesh K. Gopinath, Ph.D.

Related Article: Scientists Use CRISPR To Fight Duchenne Muscular Dystrophy

References

1. https://doi.org/10.1038/nature.2016.20988
2. https://www.nature.com/articles/d41586-020-01262-3

Author

Sahana Shankar