Structural study of AAVrh.10 Receptor and Antibody Interactions

2021 ◽  
Author(s):  
Mario Mietzsch ◽  
Jennifer C. Yu ◽  
Jane Hsi ◽  
Paul Chipman ◽  
Felix Broecker ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Binding Site ◽  
Neutralizing Antibodies ◽  
Three Dimensional ◽  
Target Cells ◽  
Adeno Associated Virus ◽  
Terminal Galactose ◽  
Gene Therapy Vectors ◽  
Important Challenge

Recombinant Adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential pre-existing neutralizing host antibodies and bind to the receptor of the target cells. Both these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction were used to map the binding site of sulfated N -Acetyllactosamine (LacNAc, previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion, that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. Importance Gene therapy vectors based on Adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, pre-existing neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.

scholarly journals Adeno-Associated Virus (AAV) Gene Delivery: Dissecting Molecular Interactions Upon Cell Entry

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1336
Author(s):  
Edward E. Large ◽  
Mark A. Silveria ◽  
Grant M. Zane ◽  
Onellah Weerakoon ◽  
Michael S. Chapman
Keyword(s):  
Gene Therapy ◽  
Molecular Interactions ◽  
Neutralizing Antibodies ◽  
Major Gene ◽  
Cell Entry ◽  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Gene Therapy Vectors ◽  
Factor Interactions

Human gene therapy has advanced from twentieth-century conception to twenty-first-century reality. The recombinant Adeno-Associated Virus (rAAV) is a major gene therapy vector. Research continues to improve rAAV safety and efficacy using a variety of AAV capsid modification strategies. Significant factors influencing rAAV transduction efficiency include neutralizing antibodies, attachment factor interactions and receptor binding. Advances in understanding the molecular interactions during rAAV cell entry combined with improved capsid modulation strategies will help guide the design and engineering of safer and more efficient rAAV gene therapy vectors.

scholarly journals Impact of Medium-Sized Extracellular Vesicles on the Transduction Efficiency of Adeno-Associated Viruses in Neuronal and Primary Astrocyte Cell Cultures

2021 ◽  
Vol 22 (8) ◽  
pp. 4221
Author(s):  
Orsolya Tünde Kovács ◽  
Eszter Soltész-Katona ◽  
Nikolett Marton ◽  
Eszter Baricza ◽  
László Hunyady ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Extracellular Vesicles ◽  
Neutralizing Antibodies ◽  
Phospholipid Bilayer ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Primary Astrocyte ◽  
Efficient Gene ◽  
Gene Therapy Vectors

(1) Adeno-associated viruses (AAV) are safe and efficient gene therapy vectors with promising results in the treatment of several diseases. Extracellular vesicles (EV) are phospholipid bilayer-surrounded structures carrying several types of lipids, proteins, and nucleic acids with the ability to cross biological barriers. EV-associated AAVs might serve as new and efficient gene therapy vectors considering that they carry the benefits of both AAVs and EVs. (2) We tested vesicle-associated AAVs and vesicles mixed with AAVs on two major cell types of the central nervous system: a neural cell line (N2A) and primary astrocyte cells. (3) In contrast to previously published in vivo observations, the extracellular vesicle packaging did not improve but, in the case of primary astrocyte cells, even inhibited the infection capacity of the AAV particles. The observed effect was not due to the inhibitory effects of the vesicles themselves, since mixing the AAVs with extracellular vesicles did not change the effectiveness. (4) Our results suggest that improvement of the in vivo efficacy of the EV-associated AAV particles is not due to the enhanced interaction between the AAV and the target cells, but most likely to the improved delivery of the AAVs through tissue barriers and to the shielding of AAVs from neutralizing antibodies.

scholarly journals Quantitation of Trace Levels of DNA Released from Disrupted Adeno-Associated Virus Gene Therapy Vectors

2021 ◽  
Author(s):  
Jared S. Bee ◽  
Kristin O'Berry ◽  
Yu (Zoe) Zhang ◽  
Megan Kuhn Phillippi ◽  
Akanksha Kaushal ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Virus Gene ◽  
Gene Therapy Vectors

In Vivo Potency Assay for Adeno-Associated Virus–Based Gene Therapy Vectors Using AAVrh.10 as an Example

2018 ◽  
Vol 29 (3) ◽  
pp. 146-155 ◽  
Author(s):  
Bishnu P. De ◽  
Alvin Chen ◽  
Christiana O. Salami ◽  
Benjamin Van de Graaf ◽  
Jonathan B. Rosenberg ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Potency Assay ◽  
Adeno Associated Virus ◽  
Gene Therapy Vectors

Adeno-associated virus as a gene therapy vector: strategies to neutralize the neutralizing antibodies

2019 ◽  
Vol 19 (3) ◽  
pp. 289-298 ◽  
Author(s):  
Majid Lotfinia ◽  
Meghdad Abdollahpour-Alitappeh ◽  
Behzad Hatami ◽  
Mohammad Reza Zali ◽  
Morteza Karimipoor
Keyword(s):  
Gene Therapy ◽  
Neutralizing Antibodies ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector

Autoimmune anemia in macaques following erythropoietin gene therapy

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3303-3304 ◽  
Author(s):  
Pierre Chenuaud ◽  
Thibaut Larcher ◽  
Joseph E. Rabinowitz ◽  
Nathalie Provost ◽  
Yan Cherel ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Therapy ◽  
Autoimmune Disease ◽  
Neutralizing Antibodies ◽  
Adeno Associated Virus ◽  
Endogenous Erythropoietin ◽  
Cynomolgus Macaques ◽  
Vector Sequences ◽  
Gene Expressing ◽  
Self Antigen

Abstract We delivered the homologous erythropoietin (Epo) cDNA driven from a doxycycline-regulated promoter via recombinant adeno-associated virus in skeletal muscle of 9 cynomolgus macaques. Upon induction, rapid supraphysiologic levels of Epo were obtained. Unexpectedly, some individuals developed a profound anemia that correlated with the appearance of neutralizing antibodies against the endogenous Epo. Both the endogenous erythropoietin and vector sequences were identical. This is the first example of the inadvertent development of an autoimmune disease in primates as a result of gene transfer of a gene expressing a self-antigen. It raises some concerns when a therapeutic protein is produced at high levels from an ectopic site. (Blood. 2004;103:3303-3304)

scholarly journals The packaging capacity of adeno-associated virus (AAV) and the potential forwild-type-plusAAV gene therapy vectors

FEBS Letters ◽  
1997 ◽  
Vol 407 (1) ◽  
pp. 78-84 ◽  
Author(s):  
Paul L. Hermonat ◽  
J.Gerald Quirk ◽  
Brian M. Bishop ◽  
Li Han
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Gene Therapy Vectors ◽  
Packaging Capacity

scholarly journals Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation

2001 ◽  
Vol 75 (16) ◽  
pp. 7662-7671 ◽  
Author(s):  
Dongsheng Duan ◽  
Ziying Yan ◽  
Yongping Yue ◽  
Wei Ding ◽  
John F. Engelhardt
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Gene Delivery ◽  
Muscle Cells ◽  
Therapeutic Benefit ◽  
Transduction Efficiency ◽  
Great Promise ◽  
Adeno Associated Virus ◽  
Muscle Gene ◽  
Duchenne's Muscular Dystrophy

ABSTRACT Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improving the safety and therapeutic benefit in clinical trials. In the present study, we compared the efficiency of gene delivery to mouse muscle cells for recombinant AAV type 2 (rAAV-2) and rAAV-2cap5 (AAV-2 genomes pseudo-packaged into AAV-5 capsids). Despite similar levels of transduction by these two vectors in undifferentiated myoblasts, pseudotyped rAAV-2cap5 demonstrated dramatically enhanced transduction in differentiated myocytes in vitro (>500-fold) and in skeletal muscle in vivo (>200-fold) compared to rAAV-2. Serotype-specific differences in transduction efficiency did not directly correlate with viral binding to muscle cells but rather appeared to involve endocytic or intracellular barriers to infection. Furthermore, application of this pseudotyped virus in a mouse model of Duchenne's muscular dystrophy also demonstrated significantly improved transduction efficiency. These findings should have a significant impact on improving rAAV-mediated gene therapy in muscle.

scholarly journals Overcoming the Cystic Fibrosis Sputum Barrier to Leading Adeno-associated Virus Gene Therapy Vectors

2014 ◽  
Vol 22 (8) ◽  
pp. 1484-1493 ◽  
Author(s):  
Benjamin S Schuster ◽  
Anthony J Kim ◽  
Joshua C Kays ◽  
Mia M Kanzawa ◽  
William B Guggino ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Virus Gene ◽  
Cystic Fibrosis Sputum ◽  
Gene Therapy Vectors

scholarly journals Examining the cross-reactivity and neutralization mechanisms of a panel of mAbs against adeno-associated virus serotypes 1 and 5

2012 ◽  
Vol 93 (2) ◽  
pp. 347-355 ◽  
Author(s):  
Carole E. Harbison ◽  
Wendy S. Weichert ◽  
Brittney L. Gurda ◽  
John A. Chiorini ◽  
Mavis Agbandje-McKenna ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Gene Delivery ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Viral Infections ◽  
Cross Reactivity ◽  
Adeno Associated Virus ◽  
Viral Capsids ◽  
Serotype 1 ◽  
Antigenic Epitopes

Neutralizing antibodies play a central role in the prevention and clearance of viral infections, but can be detrimental to the use of viral capsids for gene delivery. Antibodies present a major hurdle for ongoing clinical trials using adeno-associated viruses (AAVs); however, relatively little is known about the antigenic epitopes of most AAV serotypes or the mechanism(s) of antibody-mediated neutralization. We developed panels of AAV mAbs by repeatedly immunizing mice with AAV serotype 1 (AAV1) capsids, or by sequentially immunizing with AAV1 followed by AAV5 capsids, in order to examine the efficiency and mechanisms of antibody-mediated neutralization. The antibodies were not cross-reactive between heterologous AAV serotypes except for a low level of recognition of AAV1 capsids by the AAV5 antibodies, probably due to the initial immunization with AAV1. The neutralization efficiency of different IgGs varied and Fab fragments derived from these antibodies were generally poorly neutralizing. The antibodies appeared to display various alternative mechanisms of neutralization, which included inhibition of receptor-binding and interference with a post-attachment step.

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