Long-Read Sequencing Technology To Investigate Viral Vector Integration into Host Genome

Vector Persistence, Integration, and Reactivation and Genome Modification After Administration of Gene Therapy:

Assessing Long Term Risks Gene Therapy(GT) that modifies the host genome necessitates the mutation analysis, off-target effect, and viral vector integration into the host genome. The integrating vectors (gammaretrovirus, lentivirus,, herpesvirus capable of latency-reactivation, and genome editing products (TALEN, CRISPR) introduces the risk for delayed adverse effects. These gene therapy products pose risk by 

1) introducing the permanent change in the host genome; 

2) the potential for off-target genome modifications that can lead to aberrant gene expression, chromosomal translocation, induce malignancies, etc.; 

3) the risk for insertional mutagenesis when integrating vectors are used to deliver the gene delivery or, genome editing components, and the associated risk of tumorigenicity

Long Read Sequencing Technology for Study Vector Integration

Long-read technologies can generate continuous sequences ranging from 10 kilobases to several megabases in length directly from native DNA. With the increased accuracy, throughput, and the ability to readily traverse the most repetitive region of the human genome; the long-read sequencing technology has emerged as a popular technology for studying genomics. Long-read technology is an attractive tool to study mutation analysis and insertional mutagenesis for understanding the long term impact of gene therapy/gene editing.

Sequencing Technologies for Long Read Sequencing

• Pacific Biosciences’ (PacBio) single-molecule real-time (SMRT) sequencing 
• Oxford Nanopore Technologies’ (ONT) nanopore sequencing.

PacBio Long Read Sequencing Technology

Technical Specification

Single-Molecule, Real-Time (SMRT) Sequencing is the core technology powering our long-read sequencing platforms. This innovative approach was the first of its kind and is now a proven technology used in all fields of life science.

How SMRT Works 

Oxford Nanopore Technology (ONT) Long Reading Sequencing Technology

Technical Specification

ONT long-read sequencing technology uses linear DNA molecules rather than circular ones. These linear DNA molecules are typically one to several hundred kilobases in length but can be several megabases long.

 An enzyme motor controls the translocation of the DNA or RNA strand through the nanopore. Once the DNA or RNA has passed through, the motor protein detaches and the nanopore is ready to accept the next fragment.

DNA or RNA fragments pass through a nano-scale hole. The fluctuations in current during translocation are used to determine the DNA or RNA sequence 

How ONT Works?

Advantages of  Long Read Sequencing Technologies

Long Reads:  With reads tens of kilobases in length you can readily assemble complete genomes and sequence full-length transcripts.
High Accuracy: Sequencing free of systematic error achieves >99.999% consensus accuracy.
Uniform Coverage- No bias based on GC content means you can sequence through region inaccessible to other technologies.
Single-Molecule Resolution- Capturing sequence data from native DNA or RNA molecules enables highly accurate long reads with >99% single-molecule accuracy.
Epigenetics: With no PCR amplification step, base modifications are directly detected during sequencing.

Applications of  Long Read Sequencing Technologies

Whole Genome Sequencing: For humans, plants, animals, and microbes including de novo assembly and variant detection in complex Populations

RNA Sequencing: In-depth analysis of cDNA sequences across the entire transcriptome or targeted genes
Epigenetics: Detect DNA modifications such as methylation, histone modification tec
Targeted Sequencing: Study relevant genome targets across any regions of interest

Reference

FDA Guidance on LTFU After Administration of Gene Therapy Product

PACBIO SMRT Technology

ONT Technology

Amarsinghe et al 2020, Genome Biology

Logsdon et al 2020, Nature Review Genetics