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PLoS Biology Jul 2023Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of cell macromolecules. We engineered peptide-modified adeno-associated...
Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of cell macromolecules. We engineered peptide-modified adeno-associated virus (AAV) capsids that transduce the brain through the introduction of de novo interactions with 2 proteins expressed on the mouse blood-brain barrier (BBB), LY6A or LY6C1. The in vivo tropisms of these capsids are predictable as they are dependent on the cell- and strain-specific expression of their target protein. This approach generated hundreds of capsids with dramatically enhanced central nervous system (CNS) tropisms within a single round of screening in vitro and secondary validation in vivo thereby reducing the use of animals in comparison to conventional multi-round in vivo selections. The reproducible and quantitative data derived via this method enabled both saturation mutagenesis and machine learning (ML)-guided exploration of the capsid sequence space. Notably, during our validation process, we determined that nearly all published AAV capsids that were selected for their ability to cross the BBB in mice leverage either the LY6A or LY6C1 protein, which are not present in primates. This work demonstrates that AAV capsids can be directly targeted to specific proteins to generate potent gene delivery vectors with known mechanisms of action and predictable tropisms.
Topics: Mice; Animals; Blood-Brain Barrier; Capsid; Genetic Vectors; Central Nervous System; Capsid Proteins; Dependovirus
PubMed: 37467291
DOI: 10.1371/journal.pbio.3002112 -
Advanced Science (Weinheim,... Mar 2022Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to...
Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV-transduction. AAVs are retargeted here toward endothelial cells by coating with second-generation polyamidoamine dendrimers (G2) linked to endothelial-affine peptides (CNN). G2 AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity. Three endothelial-specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of - G2 AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti-inflammatory Annexin A1 (Anxa1) in ApoE mice (day 28). Moreover, 3) in Cas9-transgenic mice, blood pressure is monitored till day 56 after systemic application of G2 AAV9-gRNAs, targeting exons 6-10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2 AAV9-Cre transduces microvascular endothelial cells in mTmG-mice or mTmG-pigs. Functionally, G2 AAV9-S1FG mediates S1FG-leukocyte adhesion, whereas G2 AAV9-Anxa1-application reduces long-term leukocyte recruitment. Moreover, blood pressure increases in Cas9-expressing mice subjected to G2 AAV9-gRNA . Therefore, G2 AAV9 may enable gene transfer in vascular and atherosclerosis models.
Topics: Animals; Blood Pressure; Dependovirus; Endothelial Cells; Mice; Mice, Transgenic; Swine; RNA, Guide, CRISPR-Cas Systems
PubMed: 35023328
DOI: 10.1002/advs.202103867 -
Journal of Virology Jul 2023Adeno-associated virus (AAV) is a nonenveloped single-stranded DNA (ssDNA) icosahedral T=1 virus being developed as a vector for clinical gene delivery systems....
Adeno-associated virus (AAV) is a nonenveloped single-stranded DNA (ssDNA) icosahedral T=1 virus being developed as a vector for clinical gene delivery systems. Currently, there are approximately 160 AAV clinical trials, with AAV2 being the most widely studied serotype. To further understand the AAV gene delivery system, this study investigates the role of viral protein (VP) symmetry interactions on capsid assembly, genome packaging, stability, and infectivity. A total of 25 (seven 2-fold, nine 3-fold, and nine 5-fold symmetry interface) AAV2 VP variants were studied. Six 2-fold and two 5-fold variants did not assemble capsids based on native immunoblots and anti-AAV2 enzyme-linked immunosorbent assays (ELISAs). Seven of the 3-fold and seven of the 5-fold variants that assembled capsids were less stable, while the only 2-fold variant that assembled had ~2°C higher thermal stability () than recombinant wild-type AAV2 (wtAAV2). Three of the 3-fold variants (AAV2-R432A, AAV2-L510A, and N511R) had an approximately 3-log defect in genome packaging. Consistent with previous reports of the 5-fold axes, the region of the capsid is important for VP1u externalization and genome ejection, and one 5-fold variant (R404A) had a significant defect in viral infectivity. The structures of wtAAV2 packaged with a transgene (AAV2-full) and without a transgene (AAV2-empty) and one 5-fold variant (AAV2-R404A) were determined by cryo-electron microscopy and three dimensional (3D)-image reconstruction to 2.8, 2.9, and 3.6 Å resolution, respectively. These structures revealed the role of stabilizing interactions on the assembly, stability, packaging, and infectivity of the virus capsid. This study provides insight into the structural characterization and functional implications of the rational design of AAV vectors. Adeno-associated viruses (AAVs) have been shown to be useful vectors for gene therapy applications. Consequently, AAV has been approved as a biologic for the treatment of several monogenic disorders, and many additional clinical trials are ongoing. These successes have generated significant interest in all aspects of the basic biology of AAV. However, to date, there are limited data available on the importance of the capsid viral protein (VP) symmetry-related interactions required to assemble and maintain the stability of the AAV capsids and the infectivity of the AAV capsids. Characterizing the residue type and interactions at these symmetry-driven assembly interfaces of AAV2 has provided the foundation for understanding their role in AAV vectors (serotypes and engineered chimeras) and has determined the residues or regions of the capsid that can or cannot tolerate alterations.
Topics: Capsid; Dependovirus; Serogroup; Cryoelectron Microscopy; Capsid Proteins; Parvovirinae; Viral Proteins; Genetic Vectors; Virus Assembly
PubMed: 37310260
DOI: 10.1128/jvi.01772-22 -
Frontiers in Immunology 2021
Topics: Animals; Dependovirus; Genetic Therapy; Genetic Vectors; Humans
PubMed: 34992614
DOI: 10.3389/fimmu.2021.822389 -
Frontiers in Immunology 2022Recombinant adeno-associated virus (AAV) is a promising delivery vehicle for gene therapy and has been widely used in >200 clinical trials globally. There are already... (Review)
Review
Recombinant adeno-associated virus (AAV) is a promising delivery vehicle for 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.
Topics: Animals; Dependovirus; Genetic Vectors; Humans; Immunoglobulin G; Immunosuppressive Agents; Peptide Hydrolases
PubMed: 36119036
DOI: 10.3389/fimmu.2022.991832 -
The AAPS Journal Apr 2023The number of approved or investigational late phase viral vector gene therapies (GTx) has been rapidly growing. The adeno-associated virus vector (AAV) technology... (Review)
Review
The number of approved or investigational late phase viral vector gene therapies (GTx) has been rapidly growing. The adeno-associated virus vector (AAV) technology continues to be the most used GTx platform of choice. The presence of pre-existing anti-AAV immunity has been firmly established and is broadly viewed as a potential deterrent for successful AAV transduction with a possibility of negative impact on clinical efficacy and a connection to adverse events. Recommendations for the evaluation of humoral, including neutralizing and total antibody based, anti-AAV immune response have been presented elsewhere. This manuscript aims to cover considerations related to the assessment of anti-AAV cellular immune response, including review of correlations between humoral and cellular responses, potential value of cellular immunogenicity assessment, and commonly used analytical methodologies and parameters critical for monitoring assay performance. This manuscript was authored by a group of scientists involved in GTx development who represent several pharma and contract research organizations. It is our intent to provide recommendations and guidance to the industry sponsors, academic laboratories, and regulatory agencies working on AAV-based GTx viral vector modalities with the goal of achieving a more consistent approach to anti-AAV cellular immune response assessment.
Topics: Dependovirus; Genetic Therapy; Immunity, Cellular; Genetic Vectors
PubMed: 37101079
DOI: 10.1208/s12248-023-00814-5 -
Gene Nov 2021Although seen as a revolution in modern science, gene therapy has been plagued by failed clinical trials and controversial ethics in the last thirty years. Moreover,... (Review)
Review
Although seen as a revolution in modern science, gene therapy has been plagued by failed clinical trials and controversial ethics in the last thirty years. Moreover, there is no comprehensive, in-depth, high-quality analysis of global gene therapy patents. This paper proposes a method to correctly retrieve patents to address the issue and use it for the patent landscape. The results show the global patent landscape of gene therapy, with the United States dominating the field, while China has emerged as a leader in recent years. For various reasons, the EU, Korea, and Japan lag in the development of patented technologies. China has edged closer to the US in both live and indefinite patents, with the Chinese Academy of Military Medical Sciences and the Chinese Academy of Sciences leading the way, surpassing primary applicants such as the US Department of Health and Human Services, the University of California, and the University of Pennsylvania. The study also reveals four broad categories of technologies that have been extensively studied in gene therapy: basic biology of the gene and diseases, diseases being treated, gene delivery methods, and potential adverse events. What is more, Adeno-Associated Virus, Retrovirus, and Lentivirus are the most prevalent gene therapy delivery vectors after 2014. The industrial development trend revealed in this paper can provide an evidence-based basis for scientific research management and decision-making.
Topics: China; Dependovirus; European Union; Genetic Therapy; Genetic Vectors; Humans; Japan; Lentivirus; Patents as Topic; Republic of Korea; Retroviridae; United States
PubMed: 34371094
DOI: 10.1016/j.gene.2021.145889 -
Nature Communications Apr 2022Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which...
Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.
Topics: Animals; CRISPR-Cas Systems; Dependovirus; Gene Editing; Genetic Vectors; Liver; Mice; Retina; Swine
PubMed: 35414130
DOI: 10.1038/s41467-022-29550-8 -
Gene Therapy Nov 2023Despite the ups and downs in the field over three decades, the science of gene therapy has continued to advance and provide enduring treatments for increasing number of...
Despite the ups and downs in the field over three decades, the science of gene therapy has continued to advance and provide enduring treatments for increasing number of diseases. There are active clinical trials approaching a variety of inherited and acquired disorders of different organ systems. Approaches include ex vivo modification of hematologic stem cells (HSC), T lymphocytes and other immune cells, as well as in vivo delivery of genes or gene editing reagents to the relevant target cells by either local or systemic administration. In this article, we highlight success and ongoing challenges in three areas of high activity in gene therapy: inherited blood cell diseases by targeting hematopoietic stem cells, malignant disorders using immune effector cells genetically modified with chimeric antigen receptors, and ophthalmologic, neurologic, and coagulation disorders using in vivo administration of adeno-associated virus (AAV) vectors. In recent years, there have been true cures for many of these diseases, with sustained clinical benefit that exceed those from other medical approaches. Each of these treatments faces ongoing challenges, namely their high one-time costs and the complexity of manufacturing the therapeutic agents, which are biological viruses and cell products, at pharmacologic standards of quality and consistency. New models of reimbursement are needed to make these innovative treatments widely available to patients in need.
Topics: Humans; Genetic Therapy; T-Lymphocytes; Hematopoietic Stem Cells; Neoplasms; Genetic Vectors; Dependovirus; Gene Editing
PubMed: 37935854
DOI: 10.1038/s41434-023-00390-5 -
Human Gene Therapy May 2023Muscle-directed gene therapy with adeno-associated viral (AAV) vectors is undergoing clinical development for treating neuromuscular disorders and for systemic delivery... (Review)
Review
Muscle-directed gene therapy with adeno-associated viral (AAV) vectors is undergoing clinical development for treating neuromuscular disorders and for systemic delivery of therapeutic proteins. Although these approaches show considerable therapeutic benefits, they are also prone to induce potent immune responses against vector or transgene products owing to the immunogenic nature of the intramuscular delivery route, or the high doses required for systemic delivery to muscle. Major immunological concerns include antibody formation against viral capsid, complement activation, and cytotoxic T cell responses against capsid or transgene products. They can negate therapy and even lead to life-threatening immunotoxicities. Herein we review clinical observations and provide an outlook for how the field addresses these problems through a combination of vector engineering and immune modulation.
Topics: Transgenes; Injections, Intramuscular; Muscles; Immunity; Genes, Viral; Dependovirus; Genetic Vectors; Gene Transfer Techniques
PubMed: 37154743
DOI: 10.1089/hum.2023.056