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

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.

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

Clinical and Experimental Medicine ◽

10.1007/s10238-019-00557-8 ◽

2019 ◽

Vol 19 (3) ◽

pp. 289-298 ◽

Cited By ~ 9

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

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Stability of Recombinant Mosaic Adeno-Associated Virus Vector rAAV/DJ/CAG at Different Temperature Conditions

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2021.3183 ◽

2021 ◽

Vol 17 (11) ◽

pp. 2114-2124

Author(s):

Alicja Bie´nkowska-Tokarczyk ◽

Maciej Małecki

Keyword(s):

Gene Therapy ◽

Uv Light ◽

Temperature Fluctuations ◽

Effect Of Temperature ◽

Transduction Efficiency ◽

Microscopy Imaging ◽

Adeno Associated Virus ◽

Targeted Gene Delivery ◽

Gene Therapy Vectors ◽

The Stability

The nanometer size and biological characteristics of recombinant adeno-associated virus vectors (rAAV) make them particularly useful as gene therapy vectors and they have been successfully used in this role. Our latest research revealed that the rAAV/DJ/CAG mosaic vector offers highly efficient targeted gene delivery to melanoma cells metastasized to the lungs and that the transduction is temperature dependent. In order to further explore the ability of the rAAV/DJ/CAG vector to deliver highly selective transduction, this study was designed to identify the transduction stability of rAAV/DJ/CAG under various conditions. The temperatures used in this study ranged from −196 ° (liquid nitrogen) to 90 °, and the effect of temperature fluctuations (freeze-thaw, cooling-heating cycles) was also studied. This research also investigated the effects of UV radiation (ultraviolet) on the rAAV/DJ/CAG activity. Changes in the transduction efficiency were assessed via fluorescence microscopy imaging and the qPCR method. Under the test conditions, the transduction efficiency was reduced by approx. 35%, on average. High temperatures (70 °/90 °) and UV light proved to have the most detrimental impact. Changes in the stability of the rAAV/DJ/CAG structure are manifested by variations in the number of genome copies (gc) and GFP+ cells. Temperature fluctuations resulted in differences in the number of gc while maintaining a similar number of GFP+ cells, which may indicate specific changes in the rAAV/DJ/CAG structure, triggering disorders or degradation in the vector entry. This study provides interesting insights into rAAV/DJ/CAG, and the implications of these findings provide a basis for developing new protocols in cancer gene therapy.

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Cross-species permissivity: structure of a goat adeno-associated virus and its complex with the human receptor, AAVR

10.1101/2022.01.14.476406 ◽

2022 ◽

Author(s):

Edward E Large ◽

Mark A Silveria ◽

Tommi A White ◽

Michael S Chapman

Keyword(s):

Gene Therapy ◽

Conformational Changes ◽

Cell Entry ◽

Adeno Associated Virus ◽

Virus Structure ◽

Gene Therapy Vector ◽

Binding Interface ◽

Atomic Interactions ◽

Entry Receptor ◽

Protein Shell

Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV's human cell entry receptor (AAVR) contains Polycystic Kidney Disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA studies, it is shown that AAVGo.1 binds to human AAVR (huAAVR) more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of huAAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure, except for disordering of a neighboring surface loop. Only 4 of the 42 capsid protein sequence differences between AAVGo.1 and AAV5 occur at the PKD1 binding interface. These result in only minor conformational changes in AAVR, including a near rigid domain rotation with maximal displacement of the receptor by ~1 Å. A picture emerges of two classes of AAV with completely different modes of binding to the same AAVR receptor, but within each class atomic interactions are mostly conserved. IMPORTANCE Adeno-Associated Virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor huAAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.

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Structure of Adeno-Associated Virus Serotype 8, a Gene Therapy Vector

Journal of Virology ◽

10.1128/jvi.01304-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12260-12271 ◽

Cited By ~ 131

Author(s):

Hyun-Joo Nam ◽

Michael Douglas Lane ◽

Eric Padron ◽

Brittney Gurda ◽

Robert McKenna ◽

...

Keyword(s):

Amino Acids ◽

Gene Therapy ◽

Structural Features ◽

Transduction Efficiency ◽

Viral Capsid ◽

Heparin Binding ◽

Gene Therapy Vector ◽

Antibody Recognition ◽

Virus Serotype ◽

Gene Therapy Vectors

ABSTRACT Adeno-associated viruses (AAVs) are being developed as gene therapy vectors, and their efficacy could be improved by a detailed understanding of their viral capsid structures. AAV serotype 8 (AAV8) shows a significantly greater liver transduction efficiency than those of other serotypes, which has resulted in efforts to develop this virus as a gene therapy vector for hemophilia A and familial hypercholesterolemia. Pseudotyping studies show that the differential tissue tropism and transduction efficiencies exhibited by the AAVs result from differences in their capsid viral protein (VP) amino acids. Towards identifying the structural features underpinning these disparities, we report the crystal structure of the AAV8 viral capsid determined to 2.6-Å resolution. The overall topology of its common overlapping VP is similar to that previously reported for the crystal structures of AAV2 and AAV4, with an eight-stranded β-barrel and long loops between the β-strands. The most significant structural differences between AAV8 and AAV2 (the best-characterized serotype) are located on the capsid surface at protrusions surrounding the two-, three-, and fivefold axes at residues reported to control transduction efficiency and antibody recognition for AAV2. In addition, a comparison of the AAV8 and AAV2 capsid surface amino acids showed a reduced distribution of basic charge for AAV8 at the mapped AAV2 heparin sulfate receptor binding region, consistent with an observed non-heparin-binding phenotype for AAV8. Thus, this AAV8 structure provides an additional platform for mutagenesis efforts to characterize AAV capsid regions responsible for differential cellular tropism, transduction, and antigenicity for these promising gene therapy vectors.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22084221 ◽

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.

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Structural study of AAVrh.10 Receptor and Antibody Interactions

Journal of Virology ◽

10.1128/jvi.01249-21 ◽

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.

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Engineered adeno-associated virus 3 vector with reduced reactivity to serum antibodies

Scientific Reports ◽

10.1038/s41598-021-88614-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mika Ito ◽

Naomi Takino ◽

Takamasa Nomura ◽

Akihiko Kan ◽

Shin-ichi Muramatsu

Keyword(s):

Gene Therapy ◽

Neutralizing Antibodies ◽

Human Hepatocytes ◽

Transduction Efficiency ◽

Triple Mutant ◽

Promising Candidate ◽

Adeno Associated Virus ◽

Surface Loop ◽

Targeted Gene Therapy ◽

Human Primate

AbstractThe natural serotypes of adeno-associated virus (AAV) or their variants, such as AAV8 and AAV5, are commonly used as vectors in the clinical programs for liver-targeted gene therapy. While AAV8 vectors are not highly efficient at targeting primary human hepatocytes, AAV3 vectors have recently demonstrated remarkable efficiency at targeting both human and non-human primate hepatocytes. However, the presence of high levels of neutralizing antibodies (NAbs) impedes transduction into hepatocytes, representing a major obstacle to the clinical application of AAV3 vectors. Herein, we engineered the viral capsid to reduce its reactivity with pre-existing NAbs, thereby enhancing the transduction efficiency. By introducing three substitutions (S472A, S587A, and N706A) on the surface loop of AAV3B capsid protein, we generated a triple mutant AAV3 (AAV.GT5) vector with less reactivity to anti-AAV capsid NAbs. While the transduction efficiency of AAV.GT5 into human hepatocellular cell lines was similar to those of parental AAV3B, it was 50-fold higher for hepatocytes derived from humanized mice compared to AAV8 vectors. Moreover, the AAV.GT5 vector yield was similar to those of the AAV2 and AAV3B vectors. Thus, high resistance to pre-existing NAbs makes AAV.GT5 a promising candidate for future liver-targeted gene therapy clinical trials.

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