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Annual Review of Virology Sep 2019The recent market approvals of recombinant adeno-associated virus (rAAV) gene therapies in Europe and the United States are landmark achievements in the history of...
The recent market approvals of recombinant adeno-associated virus (rAAV) gene therapies in Europe and the United States are landmark achievements in the history of modern science. These approvals are also anticipated to herald the emergence of a new class of therapies for monogenic disorders, which had hitherto been considered untreatable. These events can be viewed as stemming from the convergence of several important historical trends: the study of basic virology, the development of genomic technologies, the imperative for translational impact of National Institutes of Health-funded research, and the development of economic models for commercialization of rare disease therapies. In this review, these historical trends are described and the key developments that have enabled clinical rAAV gene therapies are discussed, along with an overview of the current state of the field and future directions.
Topics: Animals; Clinical Trials as Topic; Dependovirus; Genetic Therapy; Genetic Vectors; History, 20th Century; History, 21st Century; Humans
PubMed: 31283441
DOI: 10.1146/annurev-virology-092818-015530 -
Science Translational Medicine Feb 2021Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety...
Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.
Topics: Animals; Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Immunity, Innate; Mice; Swine
PubMed: 33568518
DOI: 10.1126/scitranslmed.abd3438 -
Science Advances Sep 2022Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety....
Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors-AAVMYO2 and AAVMYO3-prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.
Topics: Animals; Bioengineering; Capsid Proteins; Dependovirus; Disease Models, Animal; Genetic Therapy; Mice; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Peptide Library
PubMed: 36129972
DOI: 10.1126/sciadv.abn4704 -
Science Translational Medicine Jun 2021Human heart failure, a leading cause of death worldwide, is a prominent example of a chronic disease that may result from poor cell renewal. The Hippo signaling pathway...
Human heart failure, a leading cause of death worldwide, is a prominent example of a chronic disease that may result from poor cell renewal. The Hippo signaling pathway is an inhibitory kinase cascade that represses adult heart muscle cell (cardiomyocyte) proliferation and renewal after myocardial infarction in genetically modified mice. Here, we investigated an adeno-associated virus 9 (AAV9)-based gene therapy to locally knock down the Hippo pathway gene Salvador () in border zone cardiomyocytes in a pig model of ischemia/reperfusion-induced myocardial infarction. Two weeks after myocardial infarction, when pigs had left ventricular systolic dysfunction, we administered AAV9--short hairpin RNA (shRNA) or a control AAV9 viral vector carrying green fluorescent protein (GFP) directly into border zone cardiomyocytes via catheter-mediated subendocardial injection. Three months after injection, pig hearts treated with a high dose of AAV9--shRNA exhibited a 14.3% improvement in ejection fraction (a measure of left ventricular systolic function), evidence of cardiomyocyte division, and reduced scar sizes compared to pigs receiving AAV9-GFP. AAV9-shRNA-treated pig hearts also displayed increased capillary density and reduced cardiomyocyte ploidy. AAV9--shRNA gene therapy was well tolerated and did not induce mortality. In addition, liver and lung pathology revealed no tumor formation. Local delivery of AAV9--shRNA gene therapy to border zone cardiomyocytes in pig hearts after myocardial infarction resulted in tissue renewal and improved function and may have utility in treating heart failure.
Topics: Animals; Dependovirus; Disease Models, Animal; Genetic Therapy; Mice; Myocardial Infarction; Myocytes, Cardiac; Signal Transduction; Swine
PubMed: 34193613
DOI: 10.1126/scitranslmed.abd6892 -
Med (New York, N.Y.) Jan 2023Adeno-associated virus (AAV) vectors are a promising vehicle for noninvasive gene delivery to the central nervous system via intravenous infusion. However, naturally...
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.
Topics: Humans; Animals; Mice; Capsid; Macaca; Genetic Vectors; Central Nervous System; Transgenes; Primates; Dependovirus
PubMed: 36417917
DOI: 10.1016/j.medj.2022.11.002 -
The Journal of Clinical Investigation Jan 2024BACKGROUNDSystemic administration of adeno-associated virus (AAV) can trigger life-threatening inflammatory responses, including thrombotic microangiopathy (TMA), acute...
BACKGROUNDSystemic administration of adeno-associated virus (AAV) can trigger life-threatening inflammatory responses, including thrombotic microangiopathy (TMA), acute kidney injury due to atypical hemolytic uremic syndrome-like complement activation, immune-mediated myocardial inflammation, and hepatic toxicity.METHODSWe describe the kinetics of immune activation following systemic AAV serotype 9 (AAV9) administration in 38 individuals following 2 distinct prophylactic immunomodulation regimens. Group 1 received corticosteroids and Group 2 received rituximab plus sirolimus in addition to steroids to prevent anti-AAV antibody formation.RESULTSGroup 1 participants had a rapid increase in immunoglobulin M (IgM) and IgG. Increase in D-dimer, decline in platelet count, and complement activation are indicative of TMA. All Group 1 participants demonstrated activation of both classical and alternative complement pathways, as indicated by depleted C4 and elevated soluble C5b-9, Ba, and Bb antigens. Group 2 patients did not have a significant change in IgM or IgG and had minimal complement activation.CONCLUSIONSThis study demonstrates that TMA in the setting of AAV gene therapy is antibody dependent (classical pathway) and amplified by the alternative complement pathway. Critical time points and interventions are identified to allow for management of immune-mediated events that impact the safety and efficacy of systemic gene therapy.
Topics: Humans; Dependovirus; Thrombotic Microangiopathies; Immunoglobulin M; Immunoglobulin G
PubMed: 37988172
DOI: 10.1172/JCI173510 -
Molecular Therapy : the Journal of the... Sep 2021Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite...
Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, delivery routes, and disease indications in a well-powered, comprehensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild-type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques' CNS after intraparenchymal (putamen) injection, or cerebrospinal fluid (CSF)-mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital-droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of rationally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been developed and made available in the public domain.
Topics: Algorithms; Animals; Capsid Proteins; Central Nervous System; DNA, Viral; Databases, Genetic; Dependovirus; Drug Administration Routes; Genetic Vectors; High-Throughput Nucleotide Sequencing; Primates; RNA, Messenger; RNA, Viral; Tissue Distribution; Transduction, Genetic
PubMed: 34298128
DOI: 10.1016/j.ymthe.2021.07.010 -
Proceedings of the National Academy of... Aug 2023Tissue macrophages, including microglia, are notoriously resistant to genetic manipulation. Here, we report the creation of Adeno-associated viruses (AAV) variants that...
Tissue macrophages, including microglia, are notoriously resistant to genetic manipulation. Here, we report the creation of Adeno-associated viruses (AAV) variants that efficiently and widely transduce microglia and tissue macrophages in vivo following intravenous delivery, with transgene expression of up to 80%. We use this technology to demonstrate manipulation of microglia gene expression and microglial ablation, thereby providing invaluable research tools for the study of these important cells.
Topics: Dependovirus; Microglia; Capsid; Transgenes; Macrophages
PubMed: 37603759
DOI: 10.1073/pnas.2302997120 -
Human Gene Therapy Aug 2023Recombinant adeno-associated virus (rAAV) has been utilized successfully for gene delivery for treatment of a variety of human diseases. To sustain the growth of...
Recombinant adeno-associated virus (rAAV) has been utilized successfully for gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (TCID) assay is an cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the TCID assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second "set threshold" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.
Topics: Humans; Dependovirus; Polymerase Chain Reaction; Genome, Viral; Real-Time Polymerase Chain Reaction
PubMed: 37276150
DOI: 10.1089/hum.2023.014 -
Science Advances Apr 2023The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the...
The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the scarcity of targets known to mediate BBB crossing. To identify novel targets, we leverage a panel of adeno-associated viruses (AAVs) previously identified through mechanism-agnostic directed evolution for improved BBB transcytosis. Screening potential cognate receptors for enhanced BBB crossing, we identify two targets: murine-restricted LY6C1 and widely conserved carbonic anhydrase IV (CA-IV). We apply AlphaFold-based in silico methods to generate capsid-receptor binding models to predict the affinity of AAVs for these identified receptors. Demonstrating how these tools can unlock target-focused engineering strategies, we create an enhanced LY6C1-binding vector, AAV-PHP.eC, that, unlike our prior PHP.eB, also works in -deficient mouse strains such as BALB/cJ. Combined with structural insights from computational modeling, the identification of primate-conserved CA-IV enables the design of more specific and potent human brain-penetrant chemicals and biologicals, including gene delivery vectors.
Topics: Mice; Humans; Animals; Blood-Brain Barrier; Carbonic Anhydrase IV; Brain; Gene Transfer Techniques; Primates; Dependovirus
PubMed: 37075114
DOI: 10.1126/sciadv.adg6618