A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency.

Manufacturing of adeno-associated viruses (AAV) for gene and cell therapy applications has increased significantly and spurred development of improved mammalian and insect cell-based production systems. We developed a baculovirus-based insect cell production system-the SGMO Helper-with a novel gene architecture and greater flexibility to modulate the expression level and content of individual Rep and Cap proteins. In addition, we incorporated modifications to the AAV6 capsid sequence that improves yield, capsid integrity, and potency. Production of recombinant AAV 6 (rAAV6) using the SGMO Helper had improved yields compared to the Bac-RepCap helper from the Kotin lab. SGMO Helper-derived rAAV6 is resistant to a previously described proteolytic cleavage unique to baculovirus-insect cell production systems and has improved capsid ratios and potency, in vitro and in vivo, compared with rAAV6 produced using Bac-RepCap. Next-generation sequencing sequence analysis demonstrated that the SGMO Helper is stable over six serial passages and rAAV6 capsids contain comparable amounts of non-vector genome DNA as rAAV6 produced using Bac-RepCap. AAV production using the SGMO Helper is scalable using bioreactors and has improved yield, capsid ratio, and in vitro potency. Our studies demonstrate that the SGMO Helper is an improved platform for AAV manufacturing to enable delivery of cutting-edge gene and cell therapies.

Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency.

OBJECTIVE: Melittin and its derivative have been developed to support effective gene delivery systems. Their ability to facilitate endosomal release enhances the delivery of nanoparticle-based gene therapy. Nevertheless, its potential application in the context of viral vectors has not received much attention. Therefore, we would like to optimize the rAAV vector by Melittin analog to improve the transduction efficiency of rAAV in liver cancer cells and explore the mechanism of Melittin analog on rAAV. METHODS: Various melittin-derived peptides were inserted into loop VIII of the capsid protein in recombinant adeno-associated virus vectors. These vectors carrying either gfp or fluc genes were subjected to quantitative polymerase chain reaction assays and transduction assays in human embryonic kidney 293 (HEK293T) cells to investigate the efficiency of vector production and gene delivery. In addition, the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice. Finally, the intricate details of the vector-mediated transduction mechanisms were explored by using pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle. RESULTS: A total of 76 melittin-related peptides were identified from existing literature. Among them, CMA-3, p5RHH and aAR3 were found to significantly inhibit transduction of rAAV2 vector crude lysate. The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV. Mechanistically, bafilomycin A1, a vacuolar endosome acidification inhibitor, completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors. Most importantly, p5RHH-rAAV8 vectors also increased hepatic transduction in vivo in C57BL/6 mice. CONCLUSION: The incorporation of melittin analogs into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression. While further modifications remain an area of interest, our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery. Please cite this article as: Meng J, He Y, Yang H, Zhou L, Wang S, Feng X, Al-shargi OY, Yu X, Zhu L, Ling, C. Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency. J Integr Med. 2024; 22(1): 72-82.

A novel capsid-XL32-derived adeno-associated virus serotype prompts retinal tropism and ameliorates choroidal neovascularization.

Gene therapy has been adapted, from the laboratory to the clinic, to treat retinopathies. In contrast to subretinal route, intravitreal delivery of AAV vectors displays the advantage of bypassing surgical injuries, but the viral particles are more prone to be nullified by the host neutralizing factors. To minimize such suppression of therapeutic effect, especially in terms of AAV2 and its derivatives, we introduced three serine-to-glycine mutations, based on the phosphorylation sites identified by mass spectrum analysis, to the XL32 capsid to generate a novel serotype named AAVYC5. Via intravitreal administration, AAVYC5 was transduced more effectively into multiple retinal layers compared with AAV2 and XL32. AAVYC5 also enabled successful delivery of anti-angiogenic molecules to rescue laser-induced choroidal neovascularization and astrogliosis in mice and non-human primates. Furthermore, we detected fewer neutralizing antibodies and binding IgG in human sera against AAVYC5 than those specific for AAV2 and XL32. Our results thus implicate this capsid-optimized AAVYC5 as a promising vector suitable for a wide population, particularly those with undesirable AAV2 seroreactivity.

Multiparametric domain insertional profiling of adeno-associated virus VP1.

Several evolved properties of adeno-associated virus (AAV), such as broad tropism and immunogenicity in humans, are barriers to AAV-based gene therapy. Most efforts to re-engineer these properties have focused on variable regions near AAV's 3-fold protrusions and capsid protein termini. To comprehensively survey AAV capsids for engineerable hotspots, we determined multiple AAV fitness phenotypes upon insertion of six structured protein domains into the entire AAV-DJ capsid protein VP1. This is the largest and most comprehensive AAV domain insertion dataset to date. Our data revealed a surprising robustness of AAV capsids to accommodate large domain insertions. Insertion permissibility depended strongly on insertion position, domain type, and measured fitness phenotype, which clustered into contiguous structural units that we could link to distinct roles in AAV assembly, stability, and infectivity. We also identified engineerable hotspots of AAV that facilitate the covalent attachment of binding scaffolds, which may represent an alternative approach to re-direct AAV tropism.

Substantially improved gene transfer to interneurons with second-generation glutamate receptor-targeted DART-AAV vectors.

BACKGROUND: Adeno-associated viral vectors (AAVs) are a widely used gene transfer platform in neuroscience. Although naturally AAV serotypes can have preferences for certain tissues, selectivity for particular cell types in the CNS does not exist. Towards interneuron targeting, capsid engineering of AAV2 including display of the designed ankyrin repeat protein (DARPin) 2K19 specific for the glutamate receptor subunit 4 (GluA4) at the N-terminus of the VP2 capsid protein has been established. The resulting AAV-VP2N is highly specific for interneurons, but exhibits rather moderate transduction efficiencies. METHODS: Two alternative insertion sites for 2K19 in the GH2/GH3 loop of capsid proteins VP1 (AAV-VP1L) or VP2 (AAV-VP2L) were exploited to yield second generation GluA4-AAVs. Having packaged reporter genes under ubiquitous promoters, the vectors were characterized for biochemical properties as well as gene delivery into cell lines and rat hippocampal slice cultures. Electrophysiological recordings monitored the functional properties of transduced cells. RESULTS: Compared to AAV-VP2N, the second-generation vectors, especially AAV-VP1L, achieved about 2-fold higher genomic titers as well as a substantially improved GluA4 binding. Improvements in gene transfer activities were 18-fold on GluA4-overexpressing A549 cells and five-fold on rat hippocampal organotypic slice cultures reaching approximately 60 % of all parvalbumin positive interneurons upon a single administration. The spiking behaviour of transduced cells was unaltered and characteristic for a heterogeneous group of interneurons. CONCLUSION: The substantially improved gene transfer activity of the second generation GluA4-targeted AAV combined with low toxicity makes this vector an attractive tool for interneuron-directed gene transfer with unrestricted promotor and transgene choice.

Modifying immune responses to adeno-associated virus vectors by capsid engineering.

De novo immune responses are considered major challenges in gene therapy. With the aim to lower innate immune responses directly in cells targeted by adeno-associated virus (AAV) vectors, we equipped the vector capsid with a peptide known to interfere with Toll-like receptor signaling. Specifically, we genetically inserted in each of the 60 AAV2 capsid subunits the myeloid differentiation primary response 88 (MyD88)-derived peptide RDVLPGT, known to block MyD88 dimerization. Inserting the peptide neither interfered with capsid assembly nor with vector production yield. The novel capsid variant, AAV2.MB453, showed superior transduction efficiency compared to AAV2 in human monocyte-derived dendritic cells and in primary human hepatocyte cultures. In line with our hypothesis, AAV2.MB453 and AAV2 differed regarding innate immune response activation in primary human cells, particularly for type I interferons. Furthermore, mice treated with AAV2.MB453 showed significantly reduced CD8+ T cell responses against the transgene product for different administration routes and against the capsid following intramuscular administration. Moreover, humoral responses against the capsid were mitigated as indicated by delayed IgG2a antibody formation and an increased NAb50. To conclude, insertion of the MyD88-derived peptide into the AAV2 capsid improved early steps of host-vector interaction and reduced innate and adaptive immune responses.

SMRT Sequencing Enables High-Throughput Identification of Novel AAVs from Capsid Shuffling and Directed Evolution.

The use of AAV capsid libraries coupled with various selection strategies has proven to be a remarkable approach for generating novel AAVs with enhanced and desired features. The inability to reliably sequence the complete capsid gene in a high-throughput manner has been the bottleneck of capsid engineering. As a result, many library strategies are confined to localized and modest alterations in the capsid, such as peptide insertions or single variable region (VR) alterations. The caveat of short reads by means of next-generation sequencing (NGS) hinders the diversity of capsid library construction, shifting the field away from whole-capsid modifications. We generated AAV capsid shuffled libraries of naturally occurring AAVs and applied directed evolution in both mice and non-human primates (NHPs), with the goal of yielding AAVs that are compatible across both species for translational applications. We recovered DNA from the tissues of injected animal and used single molecule real-time (SMRT) sequencing to identify variants enriched in the central nervous system (CNS). We provide insights and considerations for variant identification by comparing bulk tissue sequencing to that of isolated nuclei. Our work highlights the potential advantages of whole-capsid engineering, as well as indispensable methodological improvements for the analysis of recovered capsids, including the nuclei-enrichment step and SMRT sequencing.

Vector Affinity and Receptor Distribution Define Tissue-Specific Targeting in an Engineered AAV Capsid.

Unbiased in vivo selections of diverse capsid libraries can yield engineered capsids that overcome gene therapy delivery challenges like traversing the blood-brain barrier (BBB), but little is known about the parameters of capsid-receptor interactions that govern their improved activity. This hampers broader efforts in precision capsid engineering and is a practical impediment to ensuring the translatability of capsid properties between preclinical animal models and human clinical trials. In this work, we utilize the adeno-associated virus (AAV)-PHP.B-Ly6a model system to better understand the targeted delivery and BBB penetration properties of AAV vectors. This model offers a defined capsid-receptor pair that can be used to systematically define relationships between target receptor affinity and in vivo activity of engineered AAV vectors. Here, we report a high-throughput method for quantifying capsid-receptor affinity and demonstrate that direct binding assays can be used to organize a vector library into families with varied affinity for their target receptor. Our data indicate that efficient central nervous system transduction requires high levels of target receptor expression at the BBB, but it is not a requirement for receptor expression to be limited to the target tissue. We observed that enhanced receptor affinity leads to reduced transduction of off-target tissues but can negatively impact on-target cellular transduction and penetration of endothelial barriers. Together, this work provides a set of tools for defining vector-receptor affinities and demonstrates how receptor expression and affinity interact to impact the performance of engineered AAV vectors in targeting the central nervous system. IMPORTANCE Novel methods for measuring adeno-associated virus (AAV)-receptor affinities, especially in relation to vector performance in vivo, would be useful to capsid engineers as they develop AAV vectors for gene therapy applications and characterize their interactions with native or engineered receptors. Here, we use the AAV-PHP.B-Ly6a model system to assess the impact of receptor affinity on the systemic delivery and endothelial penetration properties of AAV-PHP.B vectors. We discuss how receptor affinity analysis can be used to isolate vectors with optimized properties, improve the interpretation of library selections, and ultimately translate vector activities between preclinical animal models and humans.

Capsid-modified adeno-associated virus vectors as novel vaccine platform for cancer immunotherapy.

Immunotherapy has significantly improved treatment outcomes in various cancer entities. To enhance immunogenicity and efficacy, and to further broaden its applicability, co-administration of anti-tumor vaccines is considered as a promising strategy. Here, we introduce adeno-associated virus (AAV) vectors, widely used for in vivo gene therapy, as a potent cancer vaccine platform. Our AAV vector-based vaccine combines antigen display on the capsid surface with a vector-mediated antigen overexpression targeting different components of the immune system in a unique chronological order by a single intramuscular application. Thereby, both profound and long-lasting antigen-specific T and B cell immune responses were induced. Moreover, mice receiving the vaccine were protected against tumor growth, demonstrating its efficacy in two tumor models, including the low immunogenic and aggressive B16/F10-Ova melanoma model. Remarkably, this approach was even effective in conditions of a late tumor challenge, i.e., 80 days post-vaccination, between 88% (B16/F10-Ova melanoma) and 100% (EG7 thymoma) of mice remained tumor free. Thus, decorating AAV vector particles with antigens by capsid engineering represents a potent vaccine concept for applications in cancer immunotherapy. Its modular and versatile "plug-and-play" framework enables the use of tumor antigens of choice and the easy implementation of additional modifications to enhance immunogenicity further.

AAV Engineering for Improving Tropism to the Central Nervous System.

Adeno-associated virus (AAV) is a non-pathogenic virus that mainly infects primates with the help of adenoviruses. AAV is being widely used as a delivery vector for in vivo gene therapy, as evidenced by five currently approved drugs and more than 255 clinical trials across the world. Due to its relatively low immunogenicity and toxicity, sustained efficacy, and broad tropism, AAV holds great promise for treating many indications, including central nervous system (CNS), ocular, muscular, and liver diseases. However, low delivery efficiency, especially for the CNS due to the blood-brain barrier (BBB), remains a significant challenge for more clinical application of AAV gene therapy. Thus, there is an urgent need for utilizing AAV engineering to discover next-generation capsids with improved properties, e.g., enhanced BBB penetrance, lower immunogenicity, and higher packaging efficiency. AAV engineering methods, including directed evolution, rational design, and in silico design, have been developed, resulting in the discovery of novel capsids (e.g., PhP.B, B10, PAL1A/B/C). In this review, we discuss key studies that identified engineered CNS capsids and/or established methodological improvements. Further, we also discussed important issues that need to be addressed, including cross-species translatability, cell specificity, and modular engineering to improve multiple properties simultaneously.

Single-shot AAV-vectored vaccine against SARS-CoV-2 with fast and long-lasting immunity.

Due to the insufficient long-term protection and significant efficacy reduction to new variants of current COVID-19 vaccines, the epidemic prevention and control are still challenging. Here, we employ a capsid and antigen structure engineering (CASE) strategy to manufacture an adeno-associated viral serotype 6-based vaccine (S663V-RBD), which expresses trimeric receptor binding domain (RBD) of spike protein fused with a biological adjuvant RS09. Impressively, the engineered S663V-RBD could rapidly induce a satisfactory RBD-specific IgG titer within 2 weeks and maintain the titer for more than 4 months. Compared to the licensed BBIBP-CorV (Sinopharm, China), a single-dose S663V-RBD induced more endurable and robust immune responses in mice and elicited superior neutralizing antibodies against three typical SARS-CoV-2 pseudoviruses including wild type, C.37 (Lambda) and B.1.617.2 (Delta). More interestingly, the intramuscular injection of S663V-RBD could overcome pre-existing immunity against the capsid. Given its effectiveness, the CASE-based S663V-RBD may provide a new solution for the current and next pandemic.

Systemic administration of novel engineered AAV capsids facilitates enhanced transgene expression in the macaque CNS.

BACKGROUND: Adeno-associated virus (AAV) vectors are a promising vehicle for noninvasive gene delivery to the central nervous system via intravenous infusion. However, naturally occurring serotypes have a limited ability to transduce the brain, and translating engineered capsids from mice to nonhuman primates has proved challenging. METHODS: In this study, we use an mRNA-based directed-evolution strategy in multiple strains of mice as well as a de novo selection in cynomolgus macaques to identify families of engineered vectors with increased potency in the brain and decreased tropism for the liver. FINDINGS: We compare the transgene expression capabilities of several engineered vectors and show that while some of our novel macaque-derived variants significantly outperform AAV9 in transducing the macaque brain following systemic administration, mouse-derived variants-both those identified in this study and those reported by other groups-universally do not. CONCLUSIONS: Together, the results of this work introduce a class of primate-derived engineered AAV capsids with increased therapeutic potential and highlight the critical need for using appropriate animal models to both identify and evaluate novel AAVs intended for delivery to the human central nervous system. FUNDING: This work was funded primarily through an anonymous philanthropic gift to the P.C.S. lab at the Broad Institute of MIT and Harvard and by a grant from the Howard Hughes Medical Institute to P.C.S.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo.

OBJECTIVE: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. METHODS: The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. RESULTS: rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. CONCLUSION: Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo / 中西医结合学报

OBJECTIVE@#Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy.@*METHODS@#The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.@*RESULTS@#rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.@*CONCLUSION@#Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Single-shot AAV-vectored vaccine against SARS-CoV-2 with fast and long-lasting immunity

Due to the insufficient long-term protection and significant efficacy reduction to new variants of current COVID-19 vaccines, the epidemic prevention and control are still challenging. Here, we employ a capsid and antigen structure engineering (CASE) strategy to manufacture an adeno-associated viral serotype 6-based vaccine (S663V-RBD), which expresses trimeric receptor binding domain (RBD) of spike protein fused with a biological adjuvant RS09. Impressively, the engineered S663V-RBD could rapidly induce a satisfactory RBD-specific IgG titer within 2 weeks and maintain the titer for more than 4 months. Compared to the licensed BBIBP-CorV (Sinopharm, China), a single-dose S663V-RBD induced more endurable and robust immune responses in mice and elicited superior neutralizing antibodies against three typical SARS-CoV-2 pseudoviruses including wild type, C.37 (Lambda) and B.1.617.2 (Delta). More interestingly, the intramuscular injection of S663V-RBD could overcome pre-existing immunity against the capsid. Given its effectiveness, the CASE-based S663V-RBD may provide a new solution for the current and next pandemic.

Engineering Cancer Selective Virotherapies: Are the Pieces of the Puzzle Falling into Place?

Advances in gene therapy, synthetic biology, cancer genomics, and patient-derived cancer models have expanded the repertoire of strategies for targeting human cancers using viral vectors. Novel capsids, synthetic promoters, and therapeutic payloads are being developed and assessed through approaches such as rational design, pooled library screening, and directed evolution. Ultimately, the goal is to generate precision-engineered viruses that target different facets of tumor cell biology, without compromising normal tissue and organ function. In this study, we briefly review the opportunities for engineering cancer selectivity into viral vectors at both the cell extrinsic and intrinsic level. Such stringently tumor-targeted vectors can subsequently act as platforms for the delivery of potent therapeutic transgenes, including the exciting prospect of immunotherapeutic payloads. These have the potential to eradicate nontransduced cells through stimulation of systemic anticancer immune responses, thereby side-stepping the inherent challenge of achieving gene delivery to the entire cancer cell population. We discuss the importance of using advanced primary human cellular models, such as patient-derived cultures and organoids, to enable rapid screening and triage of novel candidates using disease-relevant models. We believe this combination of improved delivery and selectivity, through novel capsids and promoters, coupled with more potent choices for the combinations of immunotherapy-based payloads seems capable of finally delivering innovative new gene therapies for oncology. Many pieces of the puzzle of how to build a virus capable of targeting human cancers appear to be falling into place.

Identification of Broadly Applicable Adeno-Associated Virus Vectors by Systematic Comparison of Commonly Used Capsid Variants In Vitro.

Adeno-associated viruses (AAVs) represent highly attractive gene therapy vectors and potent research tools for the modulation of gene expression in animal models or difficult-to-transfect cell cultures. Engineered variants, comprising chimeric, mutated, or peptide-inserted capsids, have strongly broadened the utility of AAVs by altering cellular tropism, enabling immune evasion, or increasing transduction efficiency. In this work, the performance of 50 of the most used, predominantly published, AAVs was compared on several primary cells, cell lines, and induced pluripotent stem cell-derived models from different organs, including the adipose tissue, liver, lung, brain, and eyes. To identify the most efficient capsids for each cell type, self-complementary AAVs were standardized by digital polymerase chain reaction, arrayed on 96-well plates, and screened using high-content imaging. To enable best use of the data, all results are also provided in a web app. The utility of one selected AAV variant is further exemplified in a liver fibrosis assay based on primary hepatic stellate cells, where it successfully reversed a small interfering RNA (siRNA)-induced phenotype. Most importantly, our comparative analysis revealed that a subselection of only five AAV variants (AAV2.NN, AAV9-SLRSPPS, AAV6.2, AAV6TM, and AAV1P5) enabled efficient transduction of all tested cell types and markedly outperformed other well-established capsids, such as AAV2-7m8. These findings suggest that a core panel comprising these five capsid variants is a universally applicable and sufficient tool to identify potent AAVs for gene expression modulation in cellular systems.

A versatile toolkit for overcoming AAV immunity.

Recombinant adeno-associated virus (AAV) is a promising delivery vehicle for in vivo gene therapy and has been widely used in >200 clinical trials globally. There are already several approved gene therapy products, e.g., Luxturna and Zolgensma, highlighting the remarkable potential of AAV delivery. In the past, AAV has been seen as a relatively non-immunogenic vector associated with low risk of toxicity. However, an increasing number of recent studies indicate that immune responses against AAV and transgene products could be the bottleneck of AAV gene therapy. In clinical studies, pre-existing antibodies against AAV capsids exclude many patients from receiving the treatment as there is high prevalence of antibodies among humans. Moreover, immune response could lead to loss of efficacy over time and severe toxicity, manifested as liver enzyme elevations, kidney injury, and thrombocytopenia, resulting in deaths of non-human primates and patients. Therefore, extensive efforts have been attempted to address these issues, including capsid engineering, plasmapheresis, IgG proteases, CpG depletion, empty capsid decoy, exosome encapsulation, capsid variant switch, induction of regulatory T cells, and immunosuppressants. This review will discuss these methods in detail and highlight important milestones along the way.

Fantastic AAV Gene Therapy Vectors and How to Find Them-Random Diversification, Rational Design and Machine Learning.

Parvoviruses are a diverse family of small, non-enveloped DNA viruses that infect a wide variety of species, tissues and cell types. For over half a century, their intriguing biology and pathophysiology has fueled intensive research aimed at dissecting the underlying viral and cellular mechanisms. Concurrently, their broad host specificity (tropism) has motivated efforts to develop parvoviruses as gene delivery vectors for human cancer or gene therapy applications. While the sum of preclinical and clinical data consistently demonstrates the great potential of these vectors, these findings also illustrate the importance of enhancing and restricting in vivo transgene expression in desired cell types. To this end, major progress has been made especially with vectors based on Adeno-associated virus (AAV), whose capsid is highly amenable to bioengineering, repurposing and expansion of its natural tropism. Here, we provide an overview of the state-of-the-art approaches to create new AAV variants with higher specificity and efficiency of gene transfer in on-target cells. We first review traditional and novel directed evolution approaches, including high-throughput screening of AAV capsid libraries. Next, we discuss programmable receptor-mediated targeting with a focus on two recent technologies that utilize high-affinity binders. Finally, we highlight one of the latest stratagems for rational AAV vector characterization and optimization, namely, machine learning, which promises to facilitate and accelerate the identification of next-generation, safe and precise gene delivery vehicles.

Adeno-Associated Virus Vector Design-Moving the Adeno-Associated Virus to a Bioengineered Therapeutic Nanoparticle.

Although the number of market-approved gene therapies is still low, this new class of therapeutics has become an integral part of modern medicine. The success and safety of gene therapy depend on the vectors used to deliver the therapeutic material. Adeno-associated virus (AAV) vectors have emerged as the most frequently used delivery system for in vivo gene therapy. This success was achieved with first-generation vectors, using capsids derived from natural AAV serotypes. Their broad tropism, the high seroprevalence for many of the AAV serotypes in the human population, and the high vector doses needed to transduce a sufficient number of therapy-relevant target cells are challenges that are addressed by engineering the capsid and the vector genome, improving the efficacy of these biological nanoparticles.