ASGCT Annual Meeting 2025

GMP (Good Manufacturing Practice)-grade single-guide RNA (sgRNA) is used for gene and cell therapy in clinical trials. In a seminar in 2024, the FDA has suggested that sgRNA for IND purposes should report sequence impurities with a frequency higher than 1%. Purity assessment and sequence confirmation for sgRNAs are typically performed with HPLC and Mass spectrometry. However, it is a difficult task for HPLC or Mass Spectrometry to distinguish sequence impurities of N+1 and N-1. Moreover, it is almost impossible to detect point mutations, especially when the mass difference between C and U is only 1 g/mol.

Next-generation sequencing (NGS), on the other hand, is widely used for sequence detection. Discriminating between different nucleotide bases is a routine task for NGS. However, it has its own shortcomings where reads induced by PCR bias or sequencing bias can cause the quantification to be quite inaccurate.

In this study, we have developed a semi-quantitative NGS method to analyze sequence impurities in chemical synthesized sgRNAs.

Methods: 5’rApp and T4 RNA ligase are applied to ligate the sequencing adaptors to the single-stranded sgRNA molecule. The adaptors at both ends contain UMIs (Unique Molecular Identifiers) to reduce PCR bias during amplification. Reverse Transcriptase is then used to generate a reverse complement DNA chain. The library constructed through PCR amplification is then sequenced on an Illumina platform. The data analysis process is similar to the genome sequencing except that only UMIs withplatform. The data analysis process is similar to the genome sequencing except that only UMIs with more than three reads are counted in our analysis pipeline. Tables and pie charts are drawn to display the sequence impurities and their distribution.

Results: Since there is only one reference sequence in the solution, the sequencing depth of the method can reach as high as 100,000X. To validate this method for quality control purposes, we have designed experiments to test the characteristics including specificity, linearity, accuracy, LOQ, and precision. The method was validated to detect N+1/N-1 or point mutations with an LOQ of 1%, which meets the FDA’s requirement for IND sgRNA quality control. R 2 of the linear curve of variants with frequencies between 1% and 30% is higher than 0.99. N+1/N-1 in both ends of the sequence can also be detected with this method.

Conclusions: After neutralizing biases that occur in PCR amplification and sequencing, NGS is able to meet FDA requirements and serve as a QC method for synthesized IND sgRNA.

mRNA research has experienced unprecedented growth, driving breakthroughs in fields such as vaccine development, cancer immunotherapy, regenerative medicine, and beyond. As researchers push the boundaries of mRNA-based therapeutics, the demand for efficient, high-quality plasmid DNA templates has surged. These templates are essential for in-vitro transcription (IVT) and play a critical role in ensuring the integrity and effectiveness of mRNA production. However, challenges persist, particularly in propagating plasmids containing Poly(A) tail sequences in E. coli. Researchers frequently encounter issues such as low plasmid yields, compromised Poly(A) tail integrity, and extended development timelines—challenges that significantly hinder therapeutic development and drive-up costs.

The Poly(A) tail is a vital component of plasmid templates for mRNA production, influencing transcription efficiency, mRNA stability, and downstream application success. Recognizing these pain points, GenScript has developed innovative solutions to accelerate mRNA research and streamline plasmid preparation workflows.

GenScript’s proprietary mRNA vectors, featuring prebuilt Poly(A) tail sequences, deliver consistent and optimized templates for in vitro transcription (IVT). Paired with our GenStable™ Poly(A) strain—engineered specifically for the propagation of plasmids containing Poly(A) tails—these solutions set a new standard in plasmid stability and productivity. Together, they achieve a remarkable >90% success rate in preparing intact clones with intact Poly(A) tail sequences through 8 generations, representing a 300% improvement over conventional tools, while increasing plasmid yields by over 7-fold, ensuring reliability and efficiency in mRNA production workflows.

GenScript’s advanced tools address persistent challenges in mRNA plasmid preparation, dramatically improving yield and intact Poly(A) tail success rates. These enhancements reduce time, resources, and costs, streamlining workflows and accelerating progress in mRNA research and therapeutic development.

The field of gene and cell therapy, particularly adeno-associated virus (AAV)-based therapies, is revolutionizing the treatment of genetic disorders, cancers, and rare conditions. AAV vectors are highly valued for their ability to deliver genetic material with high specificity and low immunogenicity, making them a cornerstone in therapeutic applications. However, the preparation of high-quality plasmids for AAV production presents unique challenges, particularly in maintaining the integrity of AAV inverted terminal repeat (ITR) sequences due to its repetitive and high GC-content nature.

ITRs are essential components of AAV plasmids, playing a critical role in viral genome replication, packaging, and integration. Regulatory agencies such as the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) require verification of intact ITR sequences for clinical applications, as mutations or truncations can compromise vector performance and therapeutic efficacy.

To overcome these challenges, GenScript has developed a comprehensive suite of plasmid solutions specifically designed for AAV plasmid propagation. Our proprietary GenStable™ ITR strain and AAV applied vectors with built-in ITR sequences are engineered to preserve ITR integrity, significantly enhancing sequence stability and plasmid yield. Together, these solutions achieve an impressive >95% success rate in generating ITR intact clones through 10 generations, representing a 100% improvement over conventional tools, while boosting plasmid yields by more than fourfold.

In addition, GenScript offers advanced quality control technologies to ensure precise ITR sequence analysis, addressing the limitations of traditional methods such as standard Sanger sequencing and enzyme digestion, which often fail to accurately evaluate ITR integrity. Our proprietary ITR Sanger sequencing technology provides full reads of the ITR regions with clean and reliable peaks, ensuring comprehensive verification of sequence integrity. Complementing this, ITR Nanopore sequencing delivers detailed insights into sequence purity and population heterogeneity, offering a deeper understanding of plasmid quality.

By integrating cutting-edge plasmid solutions with sequencing technologies, GenScript enables researchers to overcome challenges in AAV plasmid preparation. Our solutions streamline workflows, reduce costs, and facilitate the production of high-quality AAV vectors, accelerating the development of safe and effective gene therapies.