Long Read Sequencing for Gene Therapy

Gene therapy has emerged as a promising approach to treating a wide range of inherited diseases and is expected to help improve human health. It involves the use of viral vectors, such as adeno-associated virus (AAV), to deliver therapeutic genes to target cells, with a key step being the accurate characterization and analysis of the viral vectors used for gene delivery. CD Genomics is a leading global life sciences company providing specialized long-read sequencing solutions for AAV gene therapy to sequence the full-length AAV genome with high precision. We aim to utilize long-read sequencing to improve the discovery, design, and manufacture of AAV vectors.

Introduction to AAV Gene Therapy

Gene therapy is a method of treating hereditary diseases by adding, deleting, or correcting genetic material in the cells of a person with the disease, with the goal of altering the course of the disease. Direct delivery gene therapy is the process of extracting cells from a patient, modifying them with therapeutic genes, and injecting them back into the patient. Alternatively, the delivery method can be cell-based, wherein the isolated target cells are edited using CRISPR-Cas9 and then transferred directly back into the patient. AAV is an enveloped virus approximately 4.7 kb in size that is modified to deliver DNA to target cells. AAV vectors are often preferred because of their low virulence, dependence on other viruses for replication, broad directionality, and ability to infect both dividing and non-dividing cells. The use of methods such as qPCR or gel electrophoresis can only confirm the size distribution of AAV vectors, but cannot identify their prevalence, fragmentation, or composition. Although the next-generation sequencing has been used to perform AAV sequencing, short reads do not allow sequencing of full-length AAV molecules, which are critical for identifying truncation events and chimeras. These limitations could potentially impact the safety and efficacy of AAV gene therapy products.

Fig. 1. Targeted long-read sequencing captures CRISPR editing and AAV integration outcomes in brain. (Simpson et al., 2023)

Solutions Offering at CD Genomics

Our advanced long-read sequencing platforms, PacBio SMRT sequencing and ONT Nanopore sequencing, enable researchers to comprehensively discover, design, and validate their gene therapies. CD Genomics' AAV end-to-end workflow and flexible protocols enable the sequencing of the full-length AAV genome with high precision to identify AAV vectors potential contaminants that may affect the safety, efficacy, and cost-effectiveness of gene therapy.

Our long read sequencing solutions cover all phases of gene therapy research, including vector discovery, vector design, host integration studies, and AAV production. By employing long-read sequencing, we help researchers unravel the complexity of the AAV genome and optimize its therapeutic potential.

• AAV Vector Discovery and Design

Long read sequencing plays a critical role in the discovery and design of novel AAV coatings, which are essential for improving the efficacy and specificity of gene therapy. Our long read sequencing solutions enable researchers to perform targeted sequencing and accurately quantify the different novel AAV species present in a sample. In addition, our long-read sequencing solution iteratively improves vector design by observing the frequency of truncations, fragmentations, and other non-full-length aberrant sequences. We also utilize targeted full-length isoform sequencing (targeted Iso-Seq approach) to characterize and quantify expressed isoforms, further confirming that the transgene is capable of producing the desired mRNA transcript.

• Host Integration Studies

Understanding potential integration events of AAV vectors with the host genome is critical to ensure the safety and efficacy of gene therapy. Our long read sequencing provides a comprehensive view of integration sites, enabling researchers to analyze the frequency and structural changes of AAV insertions. This helps develop safer and more effective gene therapy strategies.

• AAV Production

Different production platforms are capable of generating different AAV genomic populations. Our long read sequencing solutions enable researchers to analyze and compare unresolved genomes, truncated species, and short DNA fragments with the flanking inverted terminal repeat (ITR) in vectors produced using a variety of technologies such as the recombinant baculovirus (rBV)/Sf9 system.

Applications of Our Long Read Sequencing Solutions for AAV Gene Therapy

• Sequence the entire AAV molecule to discover novel capsid constructs with the potential to improve tissue-specific delivery.
• Identify structural variants, truncations, chimera, and even human host material that can affect the safety and integrity of the therapeutic.

Advantages of Our Long Read Sequencing Solutions for AAV Gene Therapy

• Discover, validate, and improve AAV vector designs through full-length, highly accurate AAV sequencing.
• Confirmation of transcripts required for protein synthesis for gene therapy vector expression by full-length isoform sequencing.
• Sequence previously inaccessible genomic regions to provide base-level resolution of repeat amplifications that cause disease.
• Directly assess vector integrity without extensive preparation.
• Assess the presence of cellular impurities such as truncations, chimeras, and host genome integration.
• Characterize the AAV genome as complete, null, partial, and chimeric HiFi sequencing.

CD Genomics is committed to providing accurate, comprehensive, and high-resolution long read sequencing solutions to accelerate gene therapy research. By increasing our understanding of the genetic components of disease, scientists can improve AAV gene therapy and alter disease processes. If you have any questions, please feel free to contact us. We look forward to working with you on projects of interest.

Related Services

PacBio SMRT Sequencing
Oxford Nanopore Sequencing
Full-Length Transcript Sequencing (Iso-Seq)
Viral Whole-Genome Resequencing