长读和短读目标丰富测序方法的比较和交叉验证,以评估AAV向量集成到宿主基因组中的AAV向量集成
Mark Sheehan1, Steven W Kumpf1, Jessie Qian1
1Global Discovery, Investigative and Translational Sciences, Pfizer Inc, Groton, CT 06340, USA.
Molecular therapy. Methods & clinical development
|November 7, 2024
概括
对比短读和长读向丰富测序 (TES) 对于腺相关病毒 (AAV) 基因治疗整合分析. 长期阅读的TES发现了更少的集成地点,但揭示了载体重排,这对于安全评估至关重要.
科学领域:
- 分子生物学分子生物学
- 遗传学 遗传学 是一个
- 基因治疗 基因治疗
背景情况:
- 评估腺相关病毒 (AAV) 的宿主整合特征对于降低新型AAV基因疗法的风险至关重要.
- 针对性丰富测序 (TES) 是评估集成站点 (IS) 的一个具有成本效益的方法.
- 短读测序通常用于TES,可以量化IS和模式识别,但不能测量IS长度或重组.
研究的目的:
- 为了比较短读和长读TES用于分析AAV和lentiviral集成的能力.
- 评估读取长度对整合站点和矢量重新排列的表征的影响.
主要方法:
- 短读和长读TES方法的比较.
- 使用了AAV治疗子的样本,体外用lentiviral治疗的样本,一个稳定的细胞系和一个工程刺入的样本.
- 分析的整合位点 (IS) 数,模式,长度和重组事件.
主要成果:
- 短读和长读TES都显示了AAV和lentivirus的随机整合.
- 短读TES由于更深的覆盖,发现了更多的IS,而长读TES则在AAV治疗的动物中显示了4-40%的IS的载体重排.
- 用AAV处理的样本显示出最小的克隆扩张,与体外用lentiviral处理的样本不同.
结论:
- 短阅读和长阅读的TES都提供了关于整合场所相对数量和整体模式的相似结论.
- 长时间阅读的TES,尽管识别了较少的IS,但对于测量IS长度和检测矢量重组是必不可少的,为集成配置文件提供了更深入的见解.
- 这些发现强调了短读和长读TES在综合基因治疗安全性评估中的互补作用.
更多相关视频
09:50Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina
Published on: December 25, 2021
4.5K
09:20Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants
Published on: October 18, 2022
4.5K
相关概念视频
Improving Translational Accuracy
Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
Comparing Copy Number Variations and SNPs
Sequencing of the human genome has opened up several best-kept secrets of the genome. Scientists have identified thousands of genome variations that exist within a population. These variations can be a single nucleotide or a larger chromosomal variation.
Copy number variations or CNVs are the structural variations that cover more than 1kb of DNA sequence. The single nucleotide polymorphism (SNP), on the other hand, is a single nucleotide change or a point mutation that is found in more than 1%...
Copy number variations or CNVs are the structural variations that cover more than 1kb of DNA sequence. The single nucleotide polymorphism (SNP), on the other hand, is a single nucleotide change or a point mutation that is found in more than 1%...
Improving Translational Accuracy
Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
Estimation of k and VD of Aminoglycosides
Aminoglycosides are a class of antibiotics used to treat various bacterial infections. Clinicians must determine the elimination rate constant (k) and volume of distribution (VD) to optimize therapeutic efficacy and minimize toxicity. The k value represents the rate at which the drug is removed from the body, and the VD reflects the degree to which the drug distributes into body tissues. Accurately estimating these parameters allows healthcare professionals to tailor drug dosing to individual...
