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In an open-label phase 1 trial, gene delivery of the trophic factor neurturin via an adeno-associated type-2 vector (AAV2) was well tolerated and seemed to improve motor function in patients with advanced Parkinson's disease. We aimed to assess the safety and efficacy of AAV2-neurturin in a double-blind, phase 2 randomised trial. We did a multicentre, double-blind, sham-surgery controlled trial in patients with advanced Parkinson's disease. Patients were randomly assigned (2:1) by a central, computer generated, randomisation code to receive either AAV2-neurturin (5·4×1011 vector genomes) injected bilaterally into the putamen or sham surgery. All patients and study personnel with the exception of the neurosurgical team were masked to treatment assignment. The primary endpoint was change from baseline to 12 months in the motor subscore of the unified Parkinson's disease rating scale in the practically-defined off state. All randomly assigned patients who had at least one assessment after baseline were included in the primary analyses. This trial is registered at ClinicalTrials.gov, NCT00400634. Between December, 2006, and November, 2008, 58 patients from nine sites in the USA participated in the trial. There was no significant difference in the primary endpoint in patients treated with AAV2-neurturin compared with control individuals (difference −0·31 [SE 2·63], 95% CI −5·58 to 4·97; p=0·91). Serious adverse events occurred in 13 of 38 patients treated with AAV2-neurturin and four of 20 control individuals. Three patients in the AAV2-neurturin group and two in the sham surgery group developed tumours. Intraputaminal AAV2-neurturin is not superior to sham surgery when assessed using the UPDRS motor score at 12 months. However, the possibility of a benefit with additional targeting of the substantia nigra and longer term follow-up should be investigated in further studies. Ceregene and Michael J Fox Foundation for Parkinson's Research.

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

An adeno-associated virus (AAV) receptor protein essential for AAV2 entry into cells is identified; AAV receptor binds directly to the virus, and its ablation renders a diverse range of mammalian cell types and mice resistant to infection by AAV of multiple serotypes. A receptor for adeno-associated virus infection The recent revival of interest in gene therapy has been fueled by the availability of safer and more effective viral gene delivery methods, most notably adeno-associated virus (AAV) vectors. Jan Carette and colleagues now identify a protein that is essential for AAV entry into cells, subsequent to cell attachment. This protein, which they call AAVR, rapidly traffics from the plasma membrane to the trans -Golgi network. The authors show that the virus directly binds to AAVR and that genetic ablation of AAVR renders a diverse range of mammalian cell types and mice resistant to AAV infection. Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity 1 . They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2 ), and have been approved for treatment of lipoprotein lipase deficiency in Europe 3 . Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration 1 , 4 , yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans -Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr −/− (also known as Au040320 −/− and Kiaa0319l −/− ) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

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 KAP1 S824 and RPA2 S4/8/33 , 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 KAP1 S824 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.

Adeno-associated virus (AAV) receptor (AAVR) is an essential receptor for the entry of multiple AAV serotypes with divergent rules; however, the mechanism remains unclear. Here, we determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, revealing the molecular details by which PKD1 recognizes AAV5 and PKD2 is solely engaged with AAV1. PKD2 lies on the plateau region of the AAV1 capsid. However, the AAV5-AAVR interface is strikingly different, in which PKD1 is bound at the opposite side of the spike of the AAV5 capsid than the PKD2-interacting region of AAV1. Residues in strands F/G and the CD loop of PKD1 interact directly with AAV5, whereas residues in strands B/C/E and the BC loop of PKD2 make contact with AAV1. These findings further the understanding of the distinct mechanisms by which AAVR recognizes various AAV serotypes and provide an example of a single receptor engaging multiple viral serotypes with divergent rules. Multiple adeno-associated viruses (AAV) use the same receptor (AAVR), but the binding mode is not clear. Here, the authors determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, identify residues necessary for virus entry and compare the receptor interfaces of different AAV capsids.

Key Points HUH endonucleases contain the characteristic HUH motif (in which U represents a hydrophobic residue) and a Y motif, containing either one or two Tyr residues. HUH endonucleases catalyse breakage and joining of single-stranded DNA (ssDNA) by a unique mechanism using a Y motif Tyr to create a 5′ intermediate covalent bond with the ssDNA substrate. Many HUH endonucleases recognize and bind DNA hairpin structures in a sequence- or structure-specific way. Rep (replication) proteins are HUH endonucleases that mediate rolling circle replication in phages, plasmids and viruses. Relaxases use the HUH mechanism to catalyse plasmid replication and conjugation. Transposases are HUH endonucleases that mediate ssDNA transposition — for example, for the IS 91 family and IS 200 –IS 605 family insertion sequences, for insertion sequences with a common region (ISCRs) and for Helitrons. HUH endonucleases use the same catalytic motifs to mediate a diverse array of reactions, and this versatility has led to the widespread adoption of the HUH mechanism. Many mobile genetic elements, such as transposons, plasmids and viruses, must cleave their own DNA to effect transposition, replication or conjugation. Here, Chandler and colleagues describe the HUH endonucleases, which use a unique mechanism to cleave and rejoin single-stranded DNA in order to mobilize and disseminate such elements. HUH endonucleases are numerous and widespread in all three domains of life. The major function of these enzymes is processing a range of mobile genetic elements by catalysing cleavage and rejoining of single-stranded DNA using an active-site Tyr residue to make a transient 5′-phosphotyrosine bond with the DNA substrate. These enzymes have a key role in rolling-circle replication of plasmids and bacteriophages, in plasmid transfer, in the replication of several eukaryotic viruses and in various types of transposition. They have also been appropriated for cellular processes such as intron homing and the processing of bacterial repeated extragenic palindromes. Here, we provide an overview of these fascinating enzymes and their functions, using well-characterized examples of Rep proteins, relaxases and transposases, and we explore the molecular mechanisms used in their diverse activities.

Emotional stress is considered a severe pathogenetic factor of psychiatric disorders. However, the circuit mechanisms remain largely unclear. Using a three-chamber vicarious social defeat stress (3C-VSDS) model in mice, we here show that chronic emotional stress (CES) induces anxiety-like behavior and transient social interaction changes. Dopaminergic neurons of ventral tegmental area (VTA) are required to control this behavioral deficit. VTA dopaminergic neuron hyperactivity induced by CES is involved in the anxiety-like behavior in the innate anxiogenic environment. Chemogenetic activation of VTA dopaminergic neurons directly triggers anxiety-like behavior, while chemogenetic inhibition of these neurons promotes resilience to the CES-induced anxiety-like behavior. Moreover, VTA dopaminergic neurons receiving nucleus accumbens (NAc) projections are activated in CES mice. Bidirectional modulation of the NAc-VTA circuit mimics or reverses the CES-induced anxiety-like behavior. In conclusion, we propose that a NAc-VTA circuit critically establishes and regulates the CES-induced anxiety-like behavior. This study not only characterizes a preclinical model that is representative of the nuanced aspect of CES, but also provides insight to the circuit-level neuronal processes that underlie empathy-like behavior. Using a three-chamber vicarious social defeat stress model in mice, Qi et al. show that chronic emotional stress (CES) induced anxiety-like behavior and transient social interaction changes. Bidirectional modulation of NAc-VTA circuit mimics or reverses the CES-induced anxiety-like behavior.

Current gene therapy treatments utilizing adeno-associated virus (AAV) vectors are based on capsids of primate origin. However, pre-existing neutralizing anti-AAV antibodies, that are present in a significant portion of the population, can lead to vector inactivation and reduced therapeutic efficacy. Advances in DNA sequencing have facilitated the discovery of many AAVs from non-primate species, including isolates from pigs, which exhibit up to 50% capsid protein sequence divergence, compared to primate AAV serotypes. In this study, AAVs isolated from porcine tissues (AAVpo.1 and AAVpo.6) were selected for structural characterization due to their low capsid protein VP1 sequence identity compared to each other and to AAV9. The AAV vectors were produced via the standard triple transfection system in HEK293 cells using AAV2 rep to package AAV2-ITR vector genomes and were purified by iodixanol density gradient ultracentrifugation. The capsid structures of AAVpo.1 and AAVpo.6 were determined using cryo-electron microscopy and then compared to each other in addition to the AAV5 and AAV9 structures. Given that porcine AAVpo.6 has been reported to infect human cells and the ability to cross the blood–brain barrier, the functional characterization was focused on the identification of a potential glycan receptor utilized by the porcine capsids. Additionally, the porcine AAV capsid reactivity to human derived anti-AAV antibodies was assessed to evaluate the potential for these capsids to be used as alternative vectors for gene therapy, particularly for patients with pre-existing immunity to primate-derived AAV serotypes.

Promoterless recombinant adeno-associated virus is used without nucleases to target the human coagulation factor IX gene to the liver-expressed albumin locus in haemophilia B mice, with an on-target integration into ∼0.5% of the albumin alleles in hepatocytes; stable F9 plasma levels at 7–20% of normal were obtained, leading to normal coagulation times in treated factor-IX-deficient mice. Nuclease-free genome targeting less risky Most genome editing applications under consideration for therapeutic use require the presence of a site-specific endonuclease, but the uncontrolled presence of endonucleases in tissues can potentially cause significant adverse effects. Here, Mark Kay and colleagues describe the use of promoterless recombinant adeno-associated virus to target — without the need for nucleases — the human coagulation factor IX gene to the liver-expressed albumin locus in haemophilia B mice. The approach achieves on-target integration into about 0.5% of the albumin alleles in hepatocytes. Stable F9 plasma levels in treated factor IX deficient mice were 7–20% of normal, leading to coagulation times within the normal range. Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion 1 and oncogenesis 2 . Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy 3 . However, two safety concerns persist: endonuclease-associated adverse effects, both on-target 4 and off-target 5 , 6 ; and oncogene activation caused by promoter integration, even without nucleases 7 . Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX ( F9 ) gene to the liver-expressed mouse albumin ( Alb ) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8- F9 vector into neonatal and adult mice and achieved on-target integration into ∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7–20% of normal were obtained, and treated F9 -deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

The volume available in icosahedral virus capsids limits the size of viral genomes. To overcome this limitation, viruses have evolved strategies to increase their coding capacity by using more than one ORF while keeping the genome length constant. The assembly of virus capsids requires the coordinated interaction of a large number of subunits to generate a highly ordered structure in which the viral genome can be enclosed. To understand this process, it is essential to know which viral and nonviral components are involved in the assembly reaction. Here, we show that the adeno-associated virus (AAV) encodes a protein required for capsid formation by means of a nested, alternative ORF of the cap gene. Translation is initiated at a nonconventional translation start site, resulting in the expression of a protein with a calculated molecular weight of 23 kDa. This protein, designated assembly-activating protein (AAP), is localized in the host cell nucleolus, where AAV capsid morphogenesis occurs. AAP targets newly synthesized capsid proteins to this organelle and in addition fulfils a function in the assembly reaction itself. Sequence analysis suggests that also all other species of the genus Dependovirus encode a homologous protein in their cap gene. The arrangement of different ORFs that encode capsid proteins and an assembly factor within the same mRNA facilitates a timely coordinated expression of the components involved in the assembly process.