Double-Stranded scAAV vectors (Next Generation Vectors)
The double-stranded AAV genome is generated by mutating the terminal resolution site sequence on one side of the inverted terminal repeats (ITR), leading to the production of self-complementary AAV (scAAV). A genome <2.5 kilobases (kb; approximately half the size of the wild-type AAV genome) can therefore be packaged as a dimer in the AAV vector with the package size limit of 4.7kb.
Advantages of scAAV vector design
- A single-stranded AAV viral vector is widely used for gene therapy. Upon transduction, the single-stranded DNA must be converted into a transcriptionally active double-stranded form, which is a crucial rate-limiting step in transgene expression from an rAAV vector. With a scAAV double-stranded vector design, the synthesis of the complementary strand is not required.
- Double-stranded scAAV is less prone to DNA degradation after viral transduction, thereby increasing the number of copies of stable episomes
- Double-stranded scAAV has shown higher transgene expression levels at lower or same AAV does single-stranded AAV vector.
- A recent nonclinical study showed that a 20-fold lower dose of scAAV consisting of CRISPR guide RNA was sufficient to achieve similar insertions & deletions compared to a single-stranded AAV-gRNA vector.
- scAAV allows the administration of low dose vectors that decrease the risk of AAV-associated -serious adverse events including hepatic toxicity, renal impairment, etc.
- scAAV system has been used in several gene replacement clinical trials for the treatment of spinal muscular atrophy and limb-girdle muscular dystrophy
Mendell et al 2017 (published in NEJM) showed that a single administration of AAV9 containing the self-complementary SMN gene showed a persistent & durable expression of the SMN gene and improved motor function in subjects with Spinal Muscular Atrophy Type 1 (SMA) for up to 30 months. CHOP INTEND was used for the assessment of motor function.
Zhang et al 2020 (published in Science) evaluated the efficiency of ssAAV- and scAAV-packaged sgRNAs that target exon 45 regions of the DMD gene in C2C12 mouse myoblast cells. In this stud, SpCas9-expressing C2C12 mouse myoblasts myotubes(differentiated myoblast) were transduced with each of the AAVs. One week after viral transduction, decomposition (TIDE) analysis was used to detect INDELs within the Dmd exon 45 regions.
The study found the dose-dependent increase in total INDELs for both ssAAV- and scAAV-expressed sgRNA 1 week after viral transduction. Specifically, to reach a 10% threshold level of INDELs, scAAV (5 x e8 vg/ml) and ssAAV (1 × e10 vg/ml) were required, representing a 20-fold increase in efficiency of scAAV. To reach an intermediate level of INDELs of ~22%, 40-fold less scAAV (1.8 × e9 vg/ml) was required compared to ssAAV (7.8 × e10 vg/ml). Furthermore, a high level of INDELs (over 40%) was achieved by scAAV (7.2 × 109 vg/ml), whereas ssAAV required 5 × 1012 vg/ml, representing a 70-fold improvement in efficiency with scAAV.
This study showed the remarkable improvement of transgene expression and efficient gene editing using scAAV compared to ssAAV (Zhang et al 2020, Science).