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Porton Advanced Presents New Gene Editing Data

Microbial CRISPR/Cas systems have emerged as promising scientific tools used to modify human DNA/RNA. These systems enable researchers to examine the impact of genes on cellular behavior and treat genetic disorders [1]. In November 2023, exa-cel (CASGEVY), the first human ex-vivo gene editing treatment, was approved by the MHRA. Following the approval, it was marketed in the United States and Europe to treat patients aged 12 years and older with the following: sickle cell disease (SCD), recurrent vaso occlusive crises (VOCs), and transfusion-dependent ß-thalassemia (TDT) [2] [3] [4]. Currently, there are 77 active clinical trials registered on clinicaltrials.gov for gene editing treatments [5]. Porton Advanced provides CRISPR/Cas-based CRO services to further facilitate gene editing companies in advancing their pipelines.

Gene editing protocol design and construction of stable cell lines

The Porton Advanced technical team has optimized multiple gene editing approaches (knock-in, knock-out, and multi-loci editing) to achieve high editing efficiency in T cells, HSCs, and iPSC cell lines.

Case Study A – stable and highly efficient knock-out in T cells

In Case A, gene editing data are presented from various batches targeting different genes in T cells. After optimization, the efficiency of gene editing reaches over 95%, and cell viability reaches over 90% with a high consistency rate. Similar results were also obtained in other host cells, such as HSC.

Case Study B – stable and highly efficient knock-in in T cells

Case B demonstrates highly efficient non-viral vector insertion data based on the CRISPR/Cas system. Post optimization, the non-viral vector insertion of genes with varying lengths is achieved, and notably, an integration efficiency of 44.5% is reached for a 1.7 kb gene in T cells.

Case Study C – stable and highly efficient multi-loci editing

Case C shows a one-step multi-locus gene editing experiment in T cells using the CRISPR/Cas system. Following optimization, two target/loci and three target/loci are edited with over 80% efficiency in T cells (as per molecular testing results). On the protein level, the gene editing efficiency of double target/loci reaches 92.5%. (Please note that AAVS1 is a non-coding locus and thus cannot be tested by a FACS).

Case Study D – highly efficient gene editing optimization yields over 90% in different cell lines

In Case D, the figures on the left showcase the efficiency of gene editing and cell viability in various primary cells and cell lines. The results indicate that the gene editing capacity surpassed 90% (in some cells, it was almost 100%), and the cell viability remained largely unaffected after editing. Whereas, the figures on the right demonstrate gene editing data with pictures of iPSC cell lines before and after the editing process. In certain circumstances, we can achieve highly efficient gene editing while preserving an intact cell membrane.

CRISPR/Cas9 genome functional screening services

Porton Advanced recently launched CRISPR/Cas9 genome functional screening services in partnership with Synbio Technologies. With the help of Synbio’s Synthetic Biology Empowerment Technology platform, one-stop lentivirus packaging and genomic functional screening services are provided. These services are based on both CRISPR whole-genome sgRNA libraries and customized sgRNA libraries.


Service

Custom sgRNA production service

Porton Pharma, the parent company of Porton Advanced, offers stable, efficient, and high-quality customized sgRNA services suitable for pre-clinical research and IND filing. Porton Pharma also created a professional oligonucleotide chemical synthesis platform, equipped with a twelve-channel nucleic acid synthesizer for sgRNA synthesis.

Main Equipment

If you have any questions, please contact us via info@portonadvanced.com

References:

  • Madigan, V., Zhang, F., & Dahlman, J. E. (2023). Drug delivery systems for CRISPR-based genome editors. Nature Reviews Drug Discovery, 22(11), 875-894. https://doi.org/10.1038/s41573-023-00762-x
  • https://www.fda.gov/vaccines-blood-biologics/casgevy
  • European Commission Approves First CRISPR/Cas9 Gene-Edited Therapy, CASGEVY™ (exagamglogene autotemcel), for the Treatment of Sickle Cell Disease (SCD) and Transfusion-Dependent Beta Thalassemia (TDT) | CRISPR Therapeutics (crisprtx.com)
  • https://www.gov.uk/government/news/mhra-authorises-world-first-gene-therapy-that-aims-to-cure-sickle-cell-disease-and-transfusion-dependent-thalassemia
  • Data were from clinical trial.gov & global data,. The analysis method was referred to Hirakawa, Matthew P et al. “Gene editing and CRISPR in the clinic: current and future perspectives.” Bioscience reports vol. 40,4 (2020): BSR20200127. doi:10.1042/BSR20200127.