Surprise virus tied to pediatric hepatitis cases.

Two viruses plus a child's genetic background may explain a recent surge in the United Kingdom.

Adeno-Associated Viruses (AAV) and Host Immunity - A Race Between the Hare and the Hedgehog.

Adeno-associated viruses (AAV) have emerged as the lead vector in clinical trials and form the basis for several approved gene therapies for human diseases, mainly owing to their ability to sustain robust and long-term in vivo transgene expression, their amenability to genetic engineering of cargo and capsid, as well as their moderate toxicity and immunogenicity. Still, recent reports of fatalities in a clinical trial for a neuromuscular disease, although linked to an exceptionally high vector dose, have raised new caution about the safety of recombinant AAVs. Moreover, concerns linger about the presence of pre-existing anti-AAV antibodies in the human population, which precludes a significant percentage of patients from receiving, and benefitting from, AAV gene therapies. These concerns are exacerbated by observations of cellular immune responses and other adverse events, including detrimental off-target transgene expression in dorsal root ganglia. Here, we provide an update on our knowledge of the immunological and molecular race between AAV (the "hedgehog") and its human host (the "hare"), together with a compendium of state-of-the-art technologies which provide an advantage to AAV and which, thus, promise safer and more broadly applicable AAV gene therapies in the future.

N-terminal serine/threonine motif has diverse and important effects on behavior of multiple AAV serotypes.

Adeno-associated virus (AAV) is a promising gene therapy vector, but questions remain regarding mechanisms of basic viral functions. We previously showed that a serine/threonine (S/T) triplet motif and its flanking residues, located in the overlapping N-terminus of VP1/VP2 and highly conserved across most AAV serotypes, are critical for viral transcript production in vitro. Here we generate a panel of S/T triplet mutants in AAV serotypes 2, 4, and 9 and characterize their behaviors in vitro and in vivo using next generation sequencing. We show that S/T triplet mutations can significantly hinder some stages of transduction in a serotype-dependent manner in vitro. Interestingly, these defects are largely overcome in C57BL/6 mice, with only one mutant displaying altered behavior in vivo. Taken together, our results identify a short N-terminal capsid motif with diverse roles across several AAV serotypes which better informs engineering efforts to improve AAV as a vector for gene therapy.

Tissue and cell-type-specific transduction using rAAV vectors in lung diseases.

Gene therapy of genetically determined diseases, including some pathologies of the respiratory system, requires an efficient method for transgene delivery. Recombinant adeno-associated viral (rAAV) vectors are well studied and employed in gene therapy, as they are relatively simple and low immunogenic and able to efficiently transduce eukaryotic cells. To date, many natural and artificial (with modified capsids) AAV serotypes have been isolated, demonstrating preferential tropism toward different tissues and cells in accordance with the prevalent receptors on the cell surface. However, rAAV-mediated delivery is not strictly specific due to wide tropism of some viral serotypes. Thus, the development of the methods allowing modulating specificity of these vectors could be beneficial in some cases. This review describes various approaches for retargeting rAAV to respiratory cells, for example, using different types of capsid modifications and regulation of a transgene expression by tissue-specific promoters. Part of the review is devoted to the issues of transduction of stem and progenitor lung cells using AAV, which is a complicated task today.

Completion of the AAV Structural Atlas: Serotype Capsid Structures Reveals Clade-Specific Features.

The capsid structures of most Adeno-associated virus (AAV) serotypes, already assigned to an antigenic clade, have been previously determined. This study reports the remaining capsid structures of AAV7, AAV11, AAV12, and AAV13 determined by cryo-electron microscopy and three-dimensional image reconstruction to 2.96, 2.86, 2.54, and 2.76 Å resolution, respectively. These structures complete the structural atlas of the AAV serotype capsids. AAV7 represents the first clade D capsid structure; AAV11 and AAV12 are of a currently unassigned clade that would include AAV4; and AAV13 represents the first AAV2-AAV3 hybrid clade C capsid structure. These newly determined capsid structures all exhibit the AAV capsid features including 5-fold channels, 3-fold protrusions, 2-fold depressions, and a nucleotide binding pocket with an ordered nucleotide in genome-containing capsids. However, these structures have viral proteins that display clade-specific loop conformations. This structural characterization completes our three-dimensional library of the current AAV serotypes to provide an atlas of surface loop configurations compatible with capsid assembly and amenable for future vector engineering efforts. Derived vectors could improve gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity or receptor retargeting.

Structural characterization of a novel human adeno-associated virus capsid with neurotropic properties.

Recombinant adeno-associated viruses (rAAVs) are currently considered the safest and most reliable gene delivery vehicles for human gene therapy. Three serotype capsids, AAV1, AAV2, and AAV9, have been approved for commercial use in patients, but they may not be suitable for all therapeutic contexts. Here, we describe a novel capsid identified in a human clinical sample by high-throughput, long-read sequencing. The capsid, which we have named AAVv66, shares high sequence similarity with AAV2. We demonstrate that compared to AAV2, AAVv66 exhibits enhanced production yields, virion stability, and CNS transduction. Unique structural properties of AAVv66 visualized by cryo-EM at 2.5-Å resolution, suggest that critical residues at the three-fold protrusion and at the interface of the five-fold axis of symmetry likely contribute to the beneficial characteristics of AAVv66. Our findings underscore the potential of AAVv66 as a gene therapy vector.

Adeno-associated virus-binding antibodies detected in cats living in the Northeastern United States lack neutralizing activity.

Cats are a critical pre-clinical model for studying adeno-associated virus (AAV) vector-mediated gene therapies. A recent study has described the high prevalence of anti-AAV neutralizing antibodies among domestic cats in Switzerland. However, our knowledge of pre-existing humoral immunity against various AAV serotypes in cats is still limited. Here, we show that, although antibodies binding known AAV serotypes (AAV1 to AAV11) are prevalent in cats living in the Northeastern United States, these antibodies do not necessarily neutralize AAV infectivity. We analyzed sera from 35 client-owned, 20 feral, and 30 specific pathogen-free (SPF) cats for pre-existing AAV-binding antibodies against the 11 serotypes. Antibody prevalence was 7 to 90% with an overall median of 50%. The AAV-binding antibodies showed broad reactivities with other serotypes. Of 44 selected antibodies binding AAV2, AAV6 or AAV9, none exhibited appreciable neutralizing activities. Instead, AAV6 or AAV9-binding antibodies showed a transduction-enhancing effect. AAV6-binding antibodies were highly prevalent in SPF cats (83%), but this was primarily due to cross-reactivity with preventive vaccine-induced anti-feline panleukopenia virus antibodies. These results indicate that prevalent pre-existing immunity in cats is not necessarily inhibitory to AAV and highlight a substantial difference in the nature of AAV-binding antibodies in cats living in geographically different regions.

Novel AAV44.9-Based Vectors Display Exceptional Characteristics for Retinal Gene Therapy.

The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.

Distribution and genetic diversity of adeno-associated viruses in bats from coastal areas of Southeast China.

Bats are associated with several important zoonotic viruses from different families. One example includes adeno-associated viruses (AAVs), that are extensively detected in several animals, especially primates. To understand AAVs distribution and genetic diversity in the coastal areas of Southeast China, a total of 415 intestine samples were mostly collected from two provinces of southeast China, i.e., Zhejiang and Fujian province. Intestine samples from five bat species were collected for AAVs detection. The average prevalence rate for AAV detection among these samples was 18.6% (77 positives out of 415 samples) and ranged from 11.8 to 28.9% between the five bat species. This suggests that AAVs are widely distributed in diverse bat populations in southeast coastal areas of China. Based on the genome sequence of bat adeno-associated virus-CXC1(BtAAV-CXC1) from one AAV-positive sample, the genetic diversity of the detected AAVs were assessed and analyzed. Phylogenetic analysis revealed that BtAAV-CXC1 was comparatively distant to other major AAVs from mammals and non-mammals, with only a 52.9~64.7% nucleotide identity. However, they were phylogenetically closer to Rhinolophus sinicus bat adeno-associated virus (Rs-BtAAV1), with a 74.5% nt similarity. Partial analysis of the rep and cap overlapping open reading frame (ORF) sequences from bat AAV samples revealed 48 partial rep sequences and 23 partial cap sequences from positive samples shared 86.9 to 100% and 72.3 to 98.8% nucleotide identities among themselves, respectively. This suggests that the detected AAVs had a distinctly high genetic diversity. These findings led us to conclude that diverse AAVs may be widely distributed in bat populations from the southeast regions of China.

Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression.

Adeno-associated viral vectors have been successfully used in laboratory and clinical settings for efficient gene delivery. In these vectors, 96% of the adeno-associated virus (AAV) genome is replaced with a gene cassette of interest, leaving only the 145 bp inverted terminal repeat (ITR) sequences. These cis-elements, primarily from AAV serotype 2, are required for genome rescue, replication, packaging, and vector persistence. Previous work from our lab and others have demonstrated that the AAV ITR2 sequence has inherent transcriptional activity, which may confound intended transgene expression in therapeutic applications. Currently, AAV capsids are extensively study for vector contribution; however, a comprehensive analysis of ITR promoter activity of various AAV serotypes has not been described to date. Here, the transcriptional activity of AAV ITRs from different serotypes (1-4, 6, and 7) was compared in numerous cell lines and a mouse model. Under the conditions used here, all ITRs tested were capable of promoting transgene expression both in vitro and in vivo. However, we observed three classes of AAV ITR expression in vitro. Class I ITRs (AAV2 and 3) generated the highest level, whereas class II (AAV 4) had intermediate levels, and class III (AAV1 and 6) had the lowest levels. These expression levels were consistent across multiple cell lines. Only ITR7 demonstrated cell-type dependent transcriptional activity. In vivo, all classes had promoter activity. Next-generation sequencing revealed multiple transcriptional start sites that originated from the ITR sequence, with most arising from within the Rep binding element. The collective results demonstrate that the serotype ITR sequence may have multiple levels of influence on transgene expression cassettes independent of promoter selection.

Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle.

The adeno-associated virus (AAV) vector is an efficient tool for gene delivery in skeletal muscle. AAV-based therapies show promising results for treatment of various genetic disorders, including muscular dystrophy. These dystrophies represent a heterogeneous group of diseases affecting muscles and typically characterized by progressive skeletal muscle wasting and weakness and the development of fibrosis. The tropism of each AAV serotype has been extensively studied using systemic delivery routes, but very few studies have compared their transduction efficiency through direct intramuscular injection. Yet, in some muscular dystrophies, where only a few muscles are primarily affected, a local intramuscular injection to target these muscles would be the most appropriate route. A comprehensive comparison between different recombinant AAV (rAAV) serotypes is therefore needed. In this study, we investigated the transduction efficiency of rAAV serotypes 1-10 by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette allowing nuclear expression of LacZ to easily localize targeted cells. Detection of ß-galactosidase activity on muscle cryosections demonstrated that rAAV serotypes 1, 7, 8, 9, and 10 were more efficient than the others, with rAAV9 being the most efficient in mice. Furthermore, using a model of human muscle xenograft in immunodeficient mice, we observed that in human muscle, rAAV8 and rAAV9 had similar transduction efficiency. These findings demonstrate for the first time that the human muscle xenograft can be used to evaluate AAV-based therapeutical approaches in a human context.

Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes.

Recombinant adeno-associated virus (AAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (Tm), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye-like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the Tm of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid Tm. The study demonstrates that DSF measurement of intrinsic fluorescence signal is a simple, accurate, and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.

GPR108 Is a Highly Conserved AAV Entry Factor.

Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

Systemic Safety of a Recombinant AAV8 Vector for Human Cocaine Hydrolase Gene Therapy: A Good Laboratory Practice Preclinical Study in Mice.

Cocaine addiction continues to impose major burdens on affected individuals and broader society but is highly resistant to medical treatment or psychotherapy. This study was undertaken with the goal of Food and Drug Administration (FDA) permission for a first-in-human clinical trial of a gene therapy for treatment-seeking cocaine users to become and remain abstinent. The approach was based on intravenous administration of AAV8-hCocH, an adeno-associated viral vector encoding a modified plasma enzyme that metabolizes cocaine into harmless by-products. To assess systemic safety, we conducted "Good Laboratory Practice" (GLP) studies in cocaine-experienced and cocaine-naive mice at doses of 5E12 and 5E13 vector genomes/kg. Results showed total lack of viral vector-related adverse effects in all tests performed. Instead, mice given one injection of AAV8-hCocH and regular daily injections of cocaine had far less tissue pathology than cocaine-injected mice with no vector treatment. Biodistribution analysis showed the vector located almost exclusively in the liver. These results indicate that a liver-directed AAV8-hCocH gene transfer at reasonable dosage is safe, well tolerated, and effective. Thus, gene transfer therapy emerges as a radically new approach to treat compulsive cocaine abuse. In fact, based on these positive findings, the FDA recently accepted our latest request for investigational new drug application (IND 18579).

Chimeric Capsid Proteins Impact Transduction Efficiency of Haploid Adeno-Associated Virus Vectors.

Our previous studies have demonstrated that haploid AAV vectors made from capsids of two different serotypes induced high transduction and prevented serotype-specific antibody binding. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the VP1/VP2 of one serotype and VP3 of another compatible serotype. After systemic injection of 2 × 1010 vg of AAV vectors into mice, the haploid AAV vectors, composed of VP1/VP2 from serotypes 8 or 9, and VP3 from AAV2, displayed a two to seven-fold increase in liver transduction compared with those of parental AAV2 vectors. Furthermore, a chimeric AAV2/8 VP1/VP2 with N-terminus of VP1/VP2 from AAV2 and C-terminus (VP3 domain) from AAV8 was constructed, and produced the haploid vector 28m-2VP3 with AAV2 VP3. The haploid 28m-2VP3 vector showed a five-fold higher transduction than that of the vectors composed solely of AAV2 VPs. Remarkably, the 28m-2VP3 vectors also induced a significant increase in transgene expression compared to the vectors composed of AAV8 VP1/VP2 with AAV2 VP3. The results suggest that the difference in the VP1/VP2 N-terminal region between AAV2 and AAV8 may allow better "communication" between the VP1/VP2 N-terminus of AAV2 with its cognate VP3. Similarly, the haploid vectors, VP1/VP2 from serotypes 8 or 9 and VP3 from AAV3, achieved higher transductions in multiple tissue types beyond typical tropism compared with those of AAV3 vectors. Consistently, higher vector genome copy numbers were detected in these tissues, indicating that an incorporation of non-cognate VP1/VP2 might influence the cellular tropism of the haploid vectors. However, there was no significant difference or even decreased transductions when compared with those of parental AAV8 or AAV9 vectors. In summary, these studies provide insight into current development strategies of AAV vectors that can increase AAV transduction across multiple tissues.

Homologous Recombination-Based Genome Editing by Clade F AAVs Is Inefficient in the Absence of a Targeted DNA Break.

Adeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem and progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 and AAVS1 loci in human HSPCs and cell lines. As a control, we used AAV6, which effectively edits HSPCs but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc-finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results, therefore, do not support that clade F AAVs can perform high-efficiency genome editing in the absence of a DSB.

Divergent engagements between adeno-associated viruses with their cellular receptor AAVR.

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.

In Situ Modification of Tissue Stem and Progenitor Cell Genomes.

In vivo delivery of genome-modifying enzymes holds significant promise for therapeutic applications and functional genetic screening. Delivery to endogenous tissue stem cells, which provide an enduring source of cell replacement during homeostasis and regeneration, is of particular interest. Here, we use a sensitive Cre/lox fluorescent reporter system to test the efficiency of genome modification following in vivo transduction by adeno-associated viruses (AAVs) in tissue stem and progenitor cells. We combine immunophenotypic analyses with in vitro and in vivo assays of stem cell function to reveal effective targeting of skeletal muscle satellite cells, mesenchymal progenitors, hematopoietic stem cells, and dermal cell subsets using multiple AAV serotypes. Genome modification rates achieved through this system reached >60%, and modified cells retained key functional properties. This study establishes a powerful platform to genetically alter tissue progenitors within their physiological niche while preserving their native stem cell properties and regulatory interactions.

Safe and Sustained Expression of Human Iduronidase After Intrathecal Administration of Adeno-Associated Virus Serotype 9 in Infant Rhesus Monkeys.

Many neuropathic diseases cause early, irreversible neurologic deterioration, which warrants therapeutic intervention during the first months of life. In the case of mucopolysaccharidosis type I, a recessive lysosomal storage disorder that results from a deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), one of the most promising treatment approaches is to restore enzyme expression through gene therapy. Specifically, administering pantropic adeno-associated virus (AAV) encoding IDUA into the cerebrospinal fluid (CSF) via suboccipital administration has demonstrated remarkable efficacy in large animals. Preclinical safety studies conducted in adult nonhuman primates supported a positive risk-benefit profile of the procedure while highlighting potential subclinical toxicity to primary sensory neurons located in the dorsal root ganglia (DRG). This study investigated the long-term performance of intrathecal cervical AAV serotype 9 gene transfer of human IDUA administered to 1-month-old rhesus monkeys (N = 4) with half of the animals tolerized to the human transgene at birth via systemic administration of an AAV serotype 8 vector expressing human IDUA from the liver. Sustained expression of the transgene for almost 4 years is reported in all animals. Transduced cells were primarily pyramidal neurons in the cortex and hippocampus, Purkinje cells in the cerebellum, lower motor neurons, and DRG neurons. Both tolerized and non-tolerized animals were robust and maintained transgene expression as measured by immunohistochemical analysis of brain tissue. However, the presence of antibodies in the non-tolerized animals led to a loss of measurable levels of secreted enzyme in the CSF. These results support the safety and efficiency of treating neonatal rhesus monkeys with AAV serotype 9 gene therapy delivered into the CSF.