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The Lancet. Haematology Jul 2022A novel, engineered, liver-tropic adeno-associated virus vector expressing a hyperactive Padua factor IX (FIX) protein (BBM-H901) has been developed and is promising for...
Safety and activity of an engineered, liver-tropic adeno-associated virus vector expressing a hyperactive Padua factor IX administered with prophylactic glucocorticoids in patients with haemophilia B: a single-centre, single-arm, phase 1, pilot trial.
BACKGROUND
A novel, engineered, liver-tropic adeno-associated virus vector expressing a hyperactive Padua factor IX (FIX) protein (BBM-H901) has been developed and is promising for haemophilia B gene therapy. We aimed to explore its safety and activity in increasing FIX concentrations and reducing bleeding frequency.
METHODS
We did a single-centre, single-arm, phase 1, pilot trial evaluating the safety and activity of a single intravenous infusion of BBM-H901 at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (Tianjin, China). We enrolled adult patients with haemophilia B (aged >18 years) with baseline FIX coagulation activity (FIX:C) of less than 2 IU/dL, no FIX inhibitor, and low titre of neutralising antibodies (≤1:4) against vector capsid. Eligible participants were intravenously infused with a single dose of 5 × 10 vector genomes (vg)/kg of BBM-H901 after 1 week of prophylactic prednisone treatment (1 mg/kg per day). Primary endpoints were the incidence of treatment-related adverse events, change in alanine aminotransferase (ALT) and aspartate amino transferase (AST), and development of antibodies against vector capsid within 1 year of infusion. We report the results of the prespecified 1-year analysis following complete enrolment. The trial is registered with ClinicalTrials.gov, NCT04135300, and is complete.
FINDINGS
Between Oct 16, 2019, and Jan 13, 2021, 12 male participants were assessed, and ten Chinese participants were enrolled and infused with BBM-H901. After a median follow-up of 58 weeks (IQR 51·5-99·5), mean FIX:C reached mean 36·9 IU/dL (SD 20·5). No serious adverse events, no grade 3-4 adverse events were observed. Grade 1-2 adverse events related to BBM-H901 include pyrexia (1 [10%]) and elevation of aminotransferase(1 [10%]). No FIX inhibitors were observed. All participants developed antibodies against vector capsid after infusion. Eight (80%) participants had ALT and AST concentrations below the upper limit of normal throughout the follow-up period. Two (20%) participants had elevation of ALT and AST accompanied with decrease of FIX:C, which remained at 7 IU/dL and 11.8 IU/dL, respectively.
INTERPRETATION
This pilot study suggests that liver-tropic BBM-H901 is safe 1 year after infusion. Vector derived FIX:C concentration is sufficiently high to prevent bleeding events and minimise the need for replacement therapy in small populations with haemophilia B. These findings support further study.
FUNDING
Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences, National Key Research and Development Program of China, National Natural Science Foundation of China, Tianjin Municipal Science and Technology Commission Grant, and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences.
Topics: Adult; Dependovirus; Factor IX; Glucocorticoids; Hemophilia B; Hemorrhage; Humans; Liver; Male; Pilot Projects
PubMed: 35598604
DOI: 10.1016/S2352-3026(22)00113-2 -
Nature Reviews. Drug Discovery Mar 2021
Topics: Animals; Dependovirus; Genetic Therapy; Genetic Vectors; Humans
PubMed: 33495615
DOI: 10.1038/d41573-021-00017-7 -
Nature Communications Oct 2023Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and...
Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.
Topics: Humans; Animals; Mice; Trans-Splicing; Gene Editing; Genetic Therapy; Stargardt Disease; Genetic Vectors; Dependovirus; ATP-Binding Cassette Transporters
PubMed: 37852949
DOI: 10.1038/s41467-023-42386-0 -
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 -
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 -
Human Gene Therapy Oct 2023For successful vector-based gene therapy manufacturing, the selected adeno-associated virus (AAV) vector production system must produce vector at sufficient scale....
For successful vector-based gene therapy manufacturing, the selected adeno-associated virus (AAV) vector production system must produce vector at sufficient scale. However, concerns have arisen regarding the quality of vector produced using different systems. In this study, we compared AAV serotypes 1, 8, and 9 produced by two different systems (Sf9/baculovirus and HEK293/transfection) and purified by two separate processes. We evaluated capsid properties, including protein composition, post-translational modification, particle content profiles, and and vector potency. Vectors produced in the Sf9/baculovirus system displayed reduced incorporation of viral protein 1 and 2 into the capsid, increased capsid protein deamidation, increased empty and partially packaged particles in vector preparations, and an overall reduced potency. The differences observed were largely independent of the harvest method and purification process. These findings illustrate the need for careful consideration when choosing an AAV vector production system for clinical production.
Topics: Humans; Capsid Proteins; Capsid; HEK293 Cells; Genetic Vectors; Dependovirus
PubMed: 37597192
DOI: 10.1089/hum.2022.116 -
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