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 AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking.

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

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.

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

Background: 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.

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

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.

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

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.

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

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.

Vector-mediated l-3,4-dihydroxyphenylalanine delivery reverses motor impairments in a primate model of Parkinson's disease.

Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner.

An Improved Adeno-Associated Virus Vector for Neurological Correction of the Mouse Model of Mucopolysaccharidosis IIIA.

Patients with the lysosomal storage disease mucopolysaccharidosis IIIA (MPSIIIA) lack the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), one of the many enzymes involved in degradation of heparan sulfate. Build-up of un-degraded heparan sulfate results in severe progressive neurodegeneration for which there is currently no treatment. Experimental gene therapies based on gene addition are currently being explored.

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus.

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase.

Inhibition of somatosensory mechanotransduction by annexin A6.

Mechanically activated, slowly adapting currents in sensory neurons have been linked to noxious mechanosensation. The conotoxin NMB-1 (noxious mechanosensation blocker-1) blocks such currents and inhibits mechanical pain. Using a biotinylated form of NMB-1 in mass spectrometry analysis, we identified 67 binding proteins in sensory neurons and a sensory neuron-derived cell line, of which the top candidate was annexin A6, a membrane-associated calcium-binding protein.

AAV9 intracerebroventricular gene therapy improves lifespan, locomotor function and pathology in a mouse model of Niemann-Pick type C1 disease.

Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localized to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects.

A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency.

Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated clinical efficacy.

Adeno-associated virus Rep proteins antagonize phosphatase PP1 to counteract KAP1 repression of the latent viral genome.

Adeno-associated virus (AAV) is a small human whose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors.

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting.

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy.

Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled.

Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface.

Unlabelled: The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration.

Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.

Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ∼20 amino acids that is critical for oligomerization and function connects the two domains.

Presence of a trs-Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration.

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

Adeno-associated virus Rep represses the human integration site promoter by two pathways that are similar to those required for the regulation of the viral p5 promoter.

Unlabelled: Adeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus. However, in the absence of helper virus infection, AAV2 establishes latency by integrating its genome site specifically into PPP1R12C, a gene located on chromosome 19. This integration target site falls into one of the most gene-dense regions of the human genome, thus inviting the question as to whether the virus has evolved mechanisms to control this complex transcriptional environment in order to facilitate integration, maintain an apparently innocuous latency, and/or establish conditions that are conducive to the rescue of the integrated viral genome.

Comparative next-generation sequencing of adeno-associated virus inverted terminal repeats.

The inverted terminal repeats (ITRs) of adeno-associated virus (AAV) are notoriously difficult to sequence owing to their high GC-content (70%) and palindromic sequences that result in the formation of a very stable, 125 bp long, T-shaped hairpin structure. Here we evaluate the performance of two widely used next-generation sequencing platforms, 454 GS FLX (Roche) and MiSeq Benchtop Sequencer (Illumina), in analyzing ITRs in comparatively sequencing linear amplification-meditated PCR (LAM-PCR) amplicons derived from AAV-concatemeric structures. While our data indicate that both platforms can sequence complete ITRs, efficiencies (MiSeq: 0.

Brief report: self-organizing neuroepithelium from human pluripotent stem cells facilitates derivation of photoreceptors.

Retinitis pigmentosa, other inherited retinal diseases, and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells.

Quantification of AAV particle titers by infrared fluorescence scanning of coomassie-stained sodium dodecyl sulfate-polyacrylamide gels.

Adeno-associated virus (AAV)-based vectors have gained increasing attention as gene delivery vehicles in basic and preclinical studies as well as in human gene therapy trials. Especially for the latter two-for both safety and therapeutic efficacy reasons-a detailed characterization of all relevant parameters of the vector preparation is essential. Two important parameters that are routinely used to analyze recombinant AAV vectors are (1) the titer of viral particles containing a (recombinant) viral genome and (2) the purity of the vector preparation, most commonly assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining.

Adeno-associated virus: a key to the human genome?

Adeno-associated viruses (AAV) are widely spread throughout the human population, yet no pathology has been associated with infection. This fact, together with the availability of simple molecular techniques to alter the packaged viral genome, has made AAV a serious contender in the search for an ideal gene therapy delivery vehicle. However, our understanding of the intriguing features of this virus is far from exhausted and it is likely that the mechanisms underlying the viral lifestyle will reveal possible novel strategies that can be employed in future clinical approaches.

Transcriptional analysis of the adeno-associated virus integration site.

The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter.