quantification-adeno-associated-viral-genomes-purified-vector-samples

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of ...

standardisation-analytical-characterization-adeno-associated-virus

Standardisation in the Analytical Characterization of Adeno-Associated Virus (AAV) Vectors

Adeno-associated virus (AAV) has become a leading vector for in vivo gene therapy, with eight products currently holding marketing authorization. As the ...

KU Leuven

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.

Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors.

Wild-type adeno-associated virus (AAV) can only replicate in the presence of helper factors, which can be provided by coinfecting helper viruses such as adenoviruses and herpesviruses. The AAV genome consists of a linear, single-stranded DNA (ssDNA), which is converted into different molecular structures within the host cell. Using high-throughput sequencing, we found that herpes simplex virus 1 (HSV-1) coinfection leads to a shift in the type of AAV genome end recombination. In particular, open-end inverted terminal repeat (ITR) recombination was enhanced, whereas open-closed ITR recombination was reduced in the presence of HSV-1. We demonstrate that the HSV-1 protein ICP8 plays an essential role in HSV-1-mediated interference with AAV genome end recombination, indicating that the previously described ICP8-driven mechanism of HSV-1 genome recombination may be underlying the observed changes. We also provide evidence that additional factors, such as products of true late genes, are involved. Although HSV-1 coinfection significantly changed the type of AAV genome end recombination, no significant change in the amount of circular AAV genomes was identified.  Adeno-associated virus (AAV)-mediated gene therapy represents one of the most promising approaches for the treatment of genetic diseases. Currently, various GMP-compatible production methods can be applied to manufacture clinical-grade vector, including methods that employ helper factors derived from herpes simplex virus 1 (HSV-1). Yet, to date, we do not fully understand how HSV-1 interacts with AAV. We observed that HSV-1 modulates AAV genome ends similarly to the genome recombination events observed during HSV-1 replication and postulate that further improvements of the HSV-1 production platform may enhance packaging of the recombinant AAV particles.

Herpes Simplex Virus 1 Coinfection Modifies Adeno-associated Virus Genome End Recombination.

Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (T) of AAV2 was consistently ∼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in T, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2.

Comparative structural, biophysical, and receptor binding study of true type and wild type AAV2.

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

Rational evolution for altering the ligand preference of estrogen receptor alpha.

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states.

Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9.

Intraparenchymal convection enhanced delivery of AAV in sheep to treat Mucopolysaccharidosis IIIC.

Recombinant adeno-associated viral vectors (rAAVs) are the leading gene delivery vehicles in clinical development, yet efficient nuclear delivery remains a major barrier to effective transduction. This limitation is partly due to the incomplete understanding of rAAV's complex subcellular trafficking dynamics. Here, we establish a longitudinal confocal live-cell imaging workflow that tracks rAAV2 from 4 to 12 h post-transduction, paired with an automated 3D analysis pipeline that quantifies spatiotemporal vector distribution, cytoplasmic trafficking, nuclear accumulation, and transgene expression at single-cell resolution. We use this platform to evaluate the effects of vector dose, cell cycle progression, and the behavior of empty particles. We identify previously undescribed trafficking features associated with high transgene expression. Higher rAAV2 doses enhanced cytoplasmic trafficking and nuclear delivery, while cell cycle progression facilitated both trafficking efficiency and transgene expression. We also characterize empty rAAV2 particles, revealing distinct trafficking patterns and markedly reduced nuclear accumulation compared to genome-containing vectors. By uncovering new bottlenecks in rAAV transduction, this platform provides mechanistic insights and potential strategies to improve AAV-based gene therapy. Its generalizable design further supports broad applicability to other non-enveloped viruses.

A Longitudinal 3D Live-Cell Imaging Platform to Uncover AAV Vector-Host Dynamics at Single-Cell Resolution.

The adeno-associated virus (AAV) capsid variant known as "true type" (AAV-TT) is a capsid with enhanced central nervous system tropism and efficient retrograde transport across brain circuits. This property enables AAV-TT to transduce neurons in multiple regions projecting to the site of injection, as previously shown in rodent and sheep models. Such spreading offers the potential for broad therapeutic vector distribution to functionally connected brain areas via a single intraparenchymal administration. To assess the clinical potential of AAV-TT, we delivered a green fluorescent protein (GFP)-expressing AAV-TT vector into the left commissural putamen of two young adult . For comparison, two additional animals received AAV9-GFP. Four weeks post-injection, animals were euthanized, and whole-brain transduction patterns were analyzed using a dedicated Aiforia deep learning-based image analysis algorithm to quantify GFP+ neurons. We observed GFP expression in structures known to innervate the injection site, consistent with retrograde transport. Both AAV-TT and AAV9 showed similar overall distribution patterns, but AAV-TT demonstrated modestly enhanced neuronal transduction efficiency compared with AAV9. This study provides the first direct comparison of AAV-TT and AAV9 in the adult nonhuman primate brain and positions AAV-TT as a promising tool for disease modeling and therapeutic interventions in neurodegenerative disorders targeting anatomically distributed neural circuits.

Biodistribution of AAV-TT and AAV9 in the Nonhuman Primate Brain.

Despite the widespread use of adeno-associated virus (AAV) vectors in gene therapy, their clinical efficacy and large-scale manufacturing remain constrained by an incomplete understanding of the virus-host interactions that govern AAV gene expression and replication. Here, we identify the PP4:SMEK1/2 phosphatase complex as an important regulator of wild-type AAV replication. Binding studies show that the AAV replication proteins engage SMEK1 to negatively influence PP4 activity. Specifically, AAV Rep68 interferes with substrate recruitment to the PP4:SMEK1 complex, resulting in hyperphosphorylation of the PP4 substrates KAP1S824 and RPA2S4/8/33, which in turn enhances viral gene expression and replication. We further uncover a direct interaction between KAP1 and SMEK1, mediated by a MAPP short linear motif that binds the SMEK1 EVH1 domain. Additionally, we identify a multifunctional complex comprising PP4:SMEK1 and PP1:NIPP1 that contributes to KAP1S824 dephosphorylation. These findings reveal a previously unrecognized mechanism by which viruses subvert host phosphatases to promote replication. This mechanistic insight not only advances our understanding of AAV and phosphatase biology but also has the potential to inform strategies for enhancing AAV vector potency.

ArticleTotal : 2	Year
Publication title Cited by 1	2024
Publication title	2026

Article	Year
Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors. Scientific reports\| PubMed ID: 32034258	2020
Herpes Simplex Virus 1 Coinfection Modifies Adeno-associated Virus Genome End Recombination. Journal of virology\| PubMed ID: 33853961	2021
Comparative structural, biophysical, and receptor binding study of true type and wild type AAV2. Journal of structural biology\| PubMed ID: 34509611	2021
Rational evolution for altering the ligand preference of estrogen receptor alpha. Protein science : a publication of the Protein Society\| PubMed ID: 38511482	2024
The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states. Nucleic acids research\| PubMed ID: 33270897	2020
Intraparenchymal convection enhanced delivery of AAV in sheep to treat Mucopolysaccharidosis IIIC. Journal of translational medicine\| PubMed ID: 37407981	2023
A Longitudinal 3D Live-Cell Imaging Platform to Uncover AAV Vector-Host Dynamics at Single-Cell Resolution. International journal of molecular sciences\| PubMed ID: 41516112	2025
Biodistribution of AAV-TT and AAV9 in the Nonhuman Primate Brain. Human gene therapy\| PubMed ID: 41797378	2026
The adeno-associated virus Rep proteins target PP4:SMEK1 by preventing substrate recruitment. PLoS pathogens\| PubMed ID: 41805761	2026

Els Henckaerts

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