Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

Abstract

Short hairpin (sh)RNAs delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function in vivo and a promising gene therapy platform. Our data show that incorporation of shRNA transgenes into rAAV constructs reduces vector yield and produces a population of truncated and defective genomes. We demonstrate that sequences with hairpins or hairpin-like structures drive the generation of truncated AAV genomes through a polymerase redirection mechanism during viral genome replication. Our findings reveal the importance of genomic secondary structure when optimizing viral vector designs. We also discovered that shDNAs could be adapted to act as surrogate mutant inverted terminal repeats (mTRs), sequences that were previously thought to be required for functional self-complementary AAV vectors. The use of shDNAs as artificial mTRs opens the door to engineering a new generation of AAV vectors with improved potency, genetic stability, and safety for both preclinical studies and human gene therapy.

Conditional, inducible gene silencing in dopamine neurons reveals a sex-specific role for Rit2 GTPase in acute cocaine response and striatal function

“…However, in vivo conditional gene expression approaches, such as Cre-lox system or cell-specific promotors, are not ideal for DAergic, AAV-mediated shRNA expression. First, the hairpin-like secondary structures, inherent to shRNAs and lox sites, create AAV genome instability that heterogeneously truncates viral genomes [17]. Moreover, self-complimentary AAVs have packaging size constraints that prohibit utilizing large DAergic promoters, such as those for tyrosine hydroxylase (TH) and DAT, to conditionally drive shRNA expression [18].…”

Subgenomic satellite particle generation in recombinant AAV vectors results from DNA lesion/breakage and non-homologous end joining

“…The unique molecular configuration of SDG molecules prompted us to explore NHEJ events as the main cause of subgenomic DNA particle formation. While a DNA polymerase template switch mechanism may explain the formation of sSBG (8,9), the existence of GDM molecules, especially the large GDM which exceed the size of the parent AAV vector and have partial duplication of vector sequences in the junction (Figure 1), strongly favors NHEJ as the primary mechanism that leads to the formation of subgenomic particles.…”

“…However, all of these data have only partial genomic information on the rAAV system. In a separate study, PacBio sequencing was used to produce more long reads and covered some special categories of rAAV genomes in the vector population (8). Here we systemically characterized the molecular state of rAAV vector genomes at a single virus level.…”

Visual synchronization of two 3-variable Lotka–Volterra oscillators based on DNA strand displacement

“…1, we have. In the synchronization reaction module, it is assumed that X and Ma i attain instant equilibrium through (18) (19) and (25) (25) and (28)…”