Distinct chemical degradation pathways of AAV1 and AAV8 under thermal stress conditions revealed by analytical anion exchange chromatography and LC-MS-based peptide mapping.

Adeno-associated virus (AAV)-based gene therapy is experiencing a rapid growth in the field of medicine and holds great promise in combating a wide range of human diseases. For successful development of AAV-based products, comprehensive thermal stability studies are often required to establish storage conditions and shelf life. However, as a relatively new modality, limited studies have been reported to elucidate the chemical degradation pathways of AAV products under thermal stress conditions. In this study, we first presented an intriguing difference in charge profile shift between thermally stressed AAV8 and AAV1 capsids when analyzed by anion exchange chromatography. Subsequently, a novel and robust peptide mapping protocol was developed and applied to elucidate the underlying chemical degradation pathways of thermally stressed AAV8 and AAV1. Compared to the conventional therapeutic proteins, the unique structure of AAV capsids also led to some key differences in how modifications at specific sites may impact the overall charge properties. Finally, despite the high sequency identity, the analysis revealed that the opposite charge profile shifts between thermally stressed AAV8 and AAV1 could be mainly attributed to a single modification unique to each serotype.

Efficient Production of an Alginate Lyase in Bacillus subtilis with Combined Strategy: Vector and Host Selection, Promoter and Signal Peptide Screening, and Modification of a Translation Initiation Region.

Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis (B. subtilis), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, PnprE exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SPydhT demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis. These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis.

Construction of intelligent response gene vector based on MOF/Fe3O4/AuNRs for tumor-targeted gene delivery.

Metal-organic frameworks (MOFs) have the potential to efficiently carry cargo due to their excellent porosity and high surface area. Nevertheless, conventional MOFs and their derivatives exhibit low efficiency in transporting nucleic acids and other small molecules, as well as having poor colloidal stability. In this study, a ZIF-90 loaded with iron oxide nanoparticles and Au nanorods was prepared, and then surface-functionalized with polyethyleneimine (PEI) to create a multifunctional nanocomposite (AFZP25k) with pH, photothermal, and magnetic responsiveness. AFZP25k can condense plasmid DNA to form AFZP25k/DNA complexes, with a maximum binding efficiency of 92.85 %. DNA release assay showed significant light and pH responsiveness, with over 80 % cumulative release after 6 h of incubation. When an external magnetic field is applied, the cellular uptake efficiency in HeLa cells reached 81.51 %, with low cytotoxicity and specific distribution. In vitro transfection experiments demonstrated a gene transfection efficiency of 44.77 % in HeLa cells. Following near-infrared irradiation, the uptake efficiency and transfection efficiency of AFZP25k in HeLa cells increased by 21.3 % and 13.59 % respectively. The findings indicate the potential of AFZP25k as an efficient and targeted gene delivery vector in cancer gene therapy.

High ionic strength vector formulations enhance gene transfer to airway epithelia.

A fundamental challenge for cystic fibrosis (CF) gene therapy is ensuring sufficient transduction of airway epithelia to achieve therapeutic correction. Hypertonic saline (HTS) is frequently administered to people with CF to enhance mucus clearance. HTS transiently disrupts epithelial cell tight junctions, but its ability to improve gene transfer has not been investigated. Here, we asked if increasing the concentration of NaCl enhances the transduction efficiency of three gene therapy vectors: adenovirus, AAV, and lentiviral vectors. Vectors formulated with 3-7% NaCl exhibited markedly increased transduction for all three platforms, leading to anion channel correction in primary cultures of human CF epithelial cells and enhanced gene transfer in mouse and pig airways in vivo. The mechanism of transduction enhancement involved tonicity but not osmolarity or pH. Formulating vectors with a high ionic strength solution is a simple strategy to greatly enhance efficacy and immediately improve preclinical or clinical applications.

Chemical approaches to probe and engineer AAV vectors.

Adeno-associated virus (AAV) has emerged as the most promising vector for in vivo human gene therapy, with several therapeutic approvals in the last few years and countless more under development. Underlying this remarkable success are several attractive features that AAV offers, including lack of pathogenicity, low immunogenicity, long-term gene expression without genomic integration, the ability to infect both dividing and non-dividing cells, etc. However, the commonly used wild-type AAV capsids in therapeutic development present significant challenges, including inadequate tissue specificity and the need for large doses to attain therapeutic effectiveness, raising safety concerns. Additionally, significant preexisting adaptive immunity against most natural capsids, and the development of such anti-capsid immunity after the first treatment, represent major challenges. Strategies to engineer the AAV capsid are critically needed to address these challenges and unlock the full promise of AAV gene therapy. Chemical modification of the AAV capsid has recently emerged as a powerful new approach to engineer its properties. Unlike genetic strategies, which can be more disruptive to the delicate capsid assembly and packaging processes, "late-stage" chemical modification of the assembled capsid-whether at natural amino acid residues or site-specifically installed noncanonical amino acid residues-often enables a versatile approach to introducing new properties to the capsid. This review summarizes the significant recent progress in AAV capsid engineering strategies, with a particular focus on chemical modifications in advancing the next generation of AAV-based gene therapies.

Construction of GSH-responsive polyethyleneimine-based delivery vector for effective gene transfection.

The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified PEI with disulfide bonds showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hin vivoeven at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high glutathione intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.

Identification of new AAV vectors with enhanced blood-brain barrier penetration efficiency via organ-on-a-chip.

To overcome limitations in the generalizability and efficiency of current AAV vectors, in this current study, we constructed an AAV variant library by the insertion of random heptapeptide sequences in the receptor-binding domain of the AAV9 capsid gene. We then applied a recently developed organ-on-a-chip in vitro model of the human blood-brain barrier (BBB) to iteratively enrich for variants that efficiently cross the BBB and transduce astrocyte cells. Through multiple rounds of screening, we obtained two candidate AAV variants, AAV-M6 and AAV-M8, which showed significantly higher BBB penetration efficiency than AAV9 or AAV-PHP.eB. Quantitative PCR (qPCR) assay showed that AAV-M6 could accumulate to a 5 times higher titer, while AAV-M8 reached a 3 times higher titer, than AAV-PHP.eB in the neural chamber of the model. The transduction assay further verified that the AAV-M6 candidate vector was able to infect HA-1800 cells after crossing the BBB, suggesting it could potentially transduce brain parenchymal cells after crossing the hCMEC/D3 layer at higher efficiency than AAV-PHP.eB. Molecular simulations suggested that the human receptor proteins, LY6D and M6PR, could bind the AAV-M6 heptapeptide insertion with high affinity. This study provides two promising candidate AAV vectors and demonstrates the use of this in vitro BBB model for scalable, high-throughput screening of gene therapies. These tools can drive investigations of the mechanisms underlying BBB permeability and the cell-type specificity of virus vectors.

Lentiviral vector determinants of anion-exchange chromatography elution heterogeneity.

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).

Lipoplexes' Structure, Preparation, and Role in Managing Different Diseases.

Lipid-based vectors are becoming promising alternatives to traditional therapies over the last 2 decades specially for managing life-threatening diseases like cancer. Cationic lipids are the most prevalent non-viral vectors utilized in gene delivery. The increasing number of clinical trials about lipoplex-based gene therapy demonstrates their potential as well-established technology that can provide robust gene transfection. In this regard, this review will summarize this important point. These vectors however have a modest transfection efficiency. This limitation can be partly addressed by using functional lipids that provide a plethora of options for investigating nucleic acid-lipid interactions as well as in vitro and in vivo nucleic acid delivery for biomedical applications. Despite their lower gene transfer efficiency, lipid-based vectors such as lipoplexes have several advantages over viral ones: they are less toxic and immunogenic, can be targeted, and are simple to produce on a large scale. Researchers are actively investigating the parameters that are essential for an effective lipoplex delivery method. These include factors that influence the structure, stability, internalization, and transfection of the lipoplex. Thorough understanding of the design principles will enable synthesis of customized lipoplex formulations for life-saving therapy.

Probe capsid structure stability and dynamics of adeno-associated virus as an important viral vector for gene therapy by hydrogen-deuterium exchange-mass spectrometry.

Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.

Identifying Viral Vector Characteristics by Nanopore Sensing.

Using viral vectors as gene delivery vehicles for gene therapy necessitates their quality control. Here, we report on nanopore sensing for nondestructively inspecting genomes inside the nanoscale cargoes at the single-molecule level. Using ionic current measurements, we motion-tracked the adeno-associated virus (AAV) vectors as they translocated through a solid-state nanopore. Considering the varying contributions of the electrophoretic forces from the negatively charged internal polynucleotides of different lengths, the nanocargoes carrying longer DNA moved more slowly in the nanochannel. Moreover, ion blockage characteristics revealed their larger volume by up to approximately 3600 nm3 in proportion to the length of single-stranded DNA packaged inside, thereby allowing electrical discriminations of AAV vectors by the gene-derived physical features. The present findings can be a promising tool for the enhanced quality control of AAV products by enabling the screening of empty and intermediate vectors at the single-particle level.

Dynamic Covalent Boronate Chemistry Accelerates the Screening of Polymeric Gene Delivery Vectors via In Situ Complexation of Nucleic Acids.

Gene therapy provides exciting new therapeutic opportunities beyond the reach of traditional treatments. Despite the tremendous progress of viral vectors, their high cost, complex manufacturing, and side effects have encouraged the development of nonviral alternatives, including cationic polymers. However, these are less efficient in overcoming cellular barriers, resulting in lower transfection efficiencies. Although the exquisite structural tunability of polymers might be envisaged as a versatile tool for improving transfection, the need to fine-tune several structural parameters represents a bottleneck in current screening technologies. By taking advantage of the fast-forming and strong boronate ester bond, an archetypal example of dynamic covalent chemistry, a highly adaptable gene delivery platform is presented, in which the polycation synthesis and pDNA complexation occur in situ. The robustness of the strategy entitles the simultaneous evaluation of several structural parameters at will, enabling the accelerated screening and adaptive optimization of lead polymeric vectors using dynamic covalent libraries.

Role of Microfiltration Membrane Morphology on Nanoparticle Purification to Enhance Downstream Purification of Viral Vectors.

In the rapidly advancing realms of gene therapy and biotechnology, the efficient purification of viral vectors is pivotal for ensuring the safety and efficacy of gene therapies. This study focuses on optimizing membrane selection for viral vector purification by evaluating key properties, including porosity, thickness, pore structure, and hydrophilicity. Notably, we employed adeno-associated virus (AAV)-sized nanoparticles (20 nm), 200 nm particles, and bovine serum albumin (BSA) to model viral vector harvesting. Experimental data from constant pressure normal flow filtration (NFF) at 1 and 2 bar using four commercial flat sheet membranes revealed distinct fouling behaviors. Symmetric membranes predominantly showed internal and external pore blockage, while asymmetric membranes formed a cake layer on the surface. Hydrophilicity exhibited a positive correlation with recovery, demonstrating an enhanced recovery with increased hydrophilicity. Membranes with higher porosity and interpore connectivity showcased superior throughput, reduced operating time, and increased recovery. Asymmetric polyether sulfone (PES) membranes emerged as the optimal choice, achieving ∼100% recovery of AAV-sized particles, an ∼44% reduction in model cell debris (200 nm particles), an ∼35% decrease in BSA, and the fastest operating time of all membranes tested. This systematic investigation into fouling behaviors and membrane properties not only informs optimal conditions for viral vector recovery but also lays the groundwork for advancing membrane-based strategies in bioprocessing.

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Production of VP3-only virus-like particles of Adeno-associated virus 2 in E. coli cells.

Adeno-associated Virus (AAV) is a promising vector for gene therapy. However, few studies have focused on producing virus-like particles (VLPs) of AAV in cells, especially in E. coli. In this study, we describe a method to produce empty VP3-only VLPs of AAV2 in E. coli by co-expressing VP3 and assembly-activating protein (AAP) of AAV2. Although the yields of VLPs produced with our method were low, the VLPs were able to self-assemble in E. coli without the need of in vitro capsid assembly. The produced VLPs were characterized by immunological detection and transmission electron microscopy (TEM). In conclusion, this study demonstrated that capsid assembly of AAV2 is possible in E. coli, and E. coli may be a candidate system for production of VLPs of AAV.

Methodological Validation of Sedimentation Velocity Analytical Ultracentrifugation Method for Adeno-Associated Virus and Collaborative Calibration of System Suitability Substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.

DNA Released by Adeno-Associated Virus Strongly Alters Capsid Aggregation Kinetics in a Physiological Solution.

While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.

Isolation of Adeno-associated Viral Vectors Through a Single-step and Semi-automated Heparin Affinity Chromatography Protocol.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Fast, accurate ranking of engineered proteins by target-binding propensity using structure modeling.

Deep-learning-based methods for protein structure prediction have achieved unprecedented accuracy, yet their utility in the engineering of protein-based binders remains constrained due to a gap between the ability to predict the structures of candidate proteins and the ability toprioritize proteins by their potential to bind to a target. To bridge this gap, we introduce Automated Pairwise Peptide-Receptor Analysis for Screening Engineered proteins (APPRAISE), a method for predicting the target-binding propensity of engineered proteins. After generating structural models of engineered proteins competing for binding to a target using an established structure prediction tool such as AlphaFold-Multimer or ESMFold, APPRAISE performs a rapid (under 1 CPU second per model) scoring analysis that takes into account biophysical and geometrical constraints. As proof-of-concept cases, we demonstrate that APPRAISE can accurately classify receptor-dependent vs. receptor-independent adeno-associated viral vectors and diverse classes of engineered proteins such as miniproteins targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, nanobodies targeting a G-protein-coupled receptor, and peptides that specifically bind to transferrin receptor or programmed death-ligand 1 (PD-L1). APPRAISE is accessible through a web-based notebook interface using Google Colaboratory (https://tiny.cc/APPRAISE). With its accuracy, interpretability, and generalizability, APPRAISE promises to expand the utility of protein structure prediction and accelerate protein engineering for biomedical applications.

Cross-Species Permissivity: Structure of a Goat Adeno-Associated Virus and Its Complex with the Human Receptor AAVR.

Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV's human cell entry receptor (AAVR) contains polycystic kidney disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA (enzyme-linked immunosorbent assay) studies, it is shown that AAVGo.1 binds to human AAVR more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of AAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure. There are only minor conformational adaptations in AAVR, but there is a near-rigid rotation of PKD1 with maximal displacement of the receptor domain by ~1 Å compared to PKD1 bound to AAV5. AAVGo.1 joins AAV5 as the second member of an emerging class of AAVs whose mode of receptor-binding is completely different from other AAVs, typified by AAV2. IMPORTANCE Adeno-associated virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy, with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor AAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.