Gene therapy

The gene therapy concept started in 1960s but the first human clinical trial of gene therapy to correct a dysfunctional gene was conducted in 1990. Although early studies were hampered due to human death in clinical trials, gene therapy has only become a very hot area in recent years after tremendous achievements are made, especially in viral vector biology. The first gene therapy drug was approved in the United State in 2017 (see all the FDA approved gene therapy drugs).

Viral vectors as carriers of functional genes in in vivo or ex vivo gene therapies include adenoviruses (AD), adeno-associated viruses (AAV), herpes simplex viruses (HSV), retroviruses and lentiviruses-a type of retrovirus. Although lentiviruses are often used to introduce genes to hematopoietic stem cells and in CAR (chimeric antigen receptors)-T cell therapies, the most used viral vector for in vivo gene therapy is AAV. As of 2022, at least 137 clinical trials are going on using different serotypes of AAV, such as AAV1, AAV2, AAV2/5, AAV2/6, AAV2/8, AAV5, AAV8, AAV9 and others. The major reason why AAV is favored for gene therapy is that the virus has not been found to be associated with any human diseases. With great understanding of the biology of AAV over the years, the vector is commonly used.

AAV used for gene therapy

The prototypical AAV vector used for gene therapy contains only two long terminal repeats (LTRs) with palindromic structure, a total 145 base pairs whose sequence was known since 1980. The gene of interest is inserted between the two LTRs. The other viral genome components that are required for AAV replication, capsidation and packaging are carried in other plasmid vectors and co-transfected into the viral particle production cells such as HEK293 cells. The viral particles are released by cell lysis and harvested through a series of purification steps as the final product for gene therapy. Multiple quality control steps are necessary to make sure that viral particles meet the safety and regulation requirement.

Sanger sequencing of the LTR sequences

One of the quality control steps in making the recombinant gene construct with two LTRs flanking the gene of the interest is to check the DNA sequence of the LTR regions. The LTR structure is not stable when amplified in E. coli during the cloning process and mutations in the LTRs can negatively affect the binding of LTRs to viral proteins and therefore the viral particle production. It is critical to have homogenously correct construct for viral packaging. Because the recombinant construct is less than 5 kb, Sanger sequencing is fast and cost-effective to sequence the construct. What about NGS? NGS is a better choice to check the final purified viral particles and identify what different contaminant DNA sources are found inside or outside the capsid of the viral particle.

How can we help you with your quality control for Sanger sequencing of the LTR region?

The LTRs from AAV contains high-GC content hairpin structure and standard Sanger dye chemistry does not work well to read through the sequence. We have a whole portfolio of Sanger sequencing reagents including the high-quality SupreDye cycle sequencing kits and SupreDye dGTP cycle sequencing kits. The latter can help read through the high GC region with minimal compression. You can also read our application notes on our SupreDye cycle sequencing kits.