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.

Genetic Engineering of AAV Capsid Gene for Gene Therapy Application

2020 ◽  
Vol 20 (5) ◽  
pp. 321-332
Author(s):  
Yunbo Liu ◽  
Xu Zhang ◽  
Lin Yang
Keyword(s):  
Gene Therapy ◽  
Neutralizing Antibodies ◽  
Human Subjects ◽  
Genetic Diseases ◽  
Capsid Gene ◽  
Human Populations ◽  
Stable Gene ◽  
Efficient Gene ◽  
Gene Therapy Application

Adeno-associated virus (AAV) is a promising vector for in vivo gene therapy because of its excellent safety profile and ability to mediate stable gene expression in human subjects. However, there are still numerous challenges that need to be resolved before this gene delivery vehicle is used in clinical applications, such as the inability of AAV to effectively target specific tissues, preexisting neutralizing antibodies in human populations, and a limited AAV packaging capacity. Over the past two decades, much genetic modification work has been performed with the AAV capsid gene, resulting in a large number of variants with modified characteristics, rendering AAV a versatile vector for more efficient gene therapy applications for different genetic diseases.

scholarly journals Sort-purification of human CD34+CD90+ cells reduces target cell population and improves lentiviral transduction for gene therapy

2019 ◽  
Author(s):  
Stefan Radtke ◽  
Dnyanada Pande ◽  
Margaret Cui ◽  
Anai M. Perez ◽  
Yan-Yi Chan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Patient Specific ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Lentiviral Transduction ◽  
Human Cd34 ◽  
Hematopoietic Reconstitution ◽  
Flow Sorting

ABSTRACTHematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant and infectious diseases. We have shown in a nonhuman primate (NHP) HSC gene therapy and transplantation model that the CD34+CD90+ cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. Here we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC-enrichment strategies include the purification of CD133+ cells or CD38low/- subsets of CD34+ cells from human blood products. We directly compared these strategies to the isolation of CD90+ cells using a GMP-grade flow-sorting protocol with clinical applicability. We show that CD90+ cell selection results in 40-fold fewer target cells in comparison to CD133+ or CD38low/- CD34 subsets without compromising the engraftment potential in vivo. Single cell RNA sequencing confirmed nearly complete depletion of lineage committed progenitor cells in CD90+ fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90+ cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and genome editing products.

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.

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.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Optimized Adenovirus-Antibody Complexes Stimulate Strong Cellular and Humoral Immune Responses against an Encoded Antigen in Naïve Mice and Those with Preexisting Immunity

2011 ◽  
Vol 19 (1) ◽  
pp. 84-95 ◽  
Author(s):  
Jin Huk Choi ◽  
Joe Dekker ◽  
Stephen C. Schafer ◽  
Jobby John ◽  
Craig E. Whitfill ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Immune Responses ◽  
Neutralizing Antibodies ◽  
T Cell Responses ◽  
Transduction Efficiency ◽  
Virus Antibody ◽  
Cell Responses

ABSTRACTThe immune response to recombinant adenoviruses is the most significant impediment to their clinical use for immunization. We test the hypothesis that specific virus-antibody combinations dictate the type of immune response generated against the adenovirus and its transgene cassette under certain physiological conditions while minimizing vector-induced toxicity.In vitroandin vivoassays were used to characterize the transduction efficiency, the T and B cell responses to the encoded transgene, and the toxicity of 1 × 1011adenovirus particles mixed with different concentrations of neutralizing antibodies. Complexes formed at concentrations of 500 to 0.05 times the 50% neutralizing dose (ND50) elicited strong virus- and transgene-specific T cell responses. The 0.05-ND50formulation elicited measurable anti-transgene antibodies that were similar to those of virus alone (P= 0.07). This preparation also elicited very strong transgene-specific memory T cell responses (28.6 ± 5.2% proliferation versus 7.7 ± 1.4% for virus alone). Preexisting immunity significantly reduced all responses elicited by these formulations. Although lower concentrations (0.005 and 0.0005 ND50) of antibody did not improve cellular and humoral responses in naïve animals, they did promote strong cellular (0.005 ND50) and humoral (0.0005 ND50) responses in mice with preexisting immunity. Some virus-antibody complexes may improve the potency of adenovirus-based vaccines in naïve individuals, while others can sway the immune response in those with preexisting immunity. Additional studies with these and other virus-antibody ratios may be useful to predict and model the type of immune responses generated against a transgene in those with different levels of exposure to adenovirus.

scholarly journals Impact of Vital Dyes on Cell Viability and Transduction Efficiency of AAV Vectors Used in Retinal Gene Therapy Surgery: An In Vitro and In Vivo Analysis

2017 ◽  
Vol 6 (4) ◽  
pp. 4 ◽  
Author(s):  
Anna P. Salvetti ◽  
Maria I. Patrício ◽  
Alun R. Barnard ◽  
Harry O. Orlans ◽  
Doron G. Hickey ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Viability ◽  
Transduction Efficiency ◽  
Vital Dyes ◽  
In Vivo Analysis ◽  
Retinal Gene Therapy

scholarly journals Vectored Immunotherapeutics for Infectious Diseases: Can rAAVs Be The Game Changers for Fighting Transmissible Pathogens?

2021 ◽  
Vol 12 ◽  
Author(s):  
Wei Zhan ◽  
Manish Muhuri ◽  
Phillip W. L. Tai ◽  
Guangping Gao
Keyword(s):  
Infectious Diseases ◽  
Neutralizing Antibodies ◽  
Viral Vectors ◽  
De Novo ◽  
Genomic Variation ◽  
Transduction Efficiency ◽  
Foreign Proteins ◽  
Repeated Dosing

Conventional vaccinations and immunotherapies have encountered major roadblocks in preventing infectious diseases like HIV, influenza, and malaria. These challenges are due to the high genomic variation and immunomodulatory mechanisms inherent to these diseases. Passive transfer of broadly neutralizing antibodies may offer partial protection, but these treatments require repeated dosing. Some recombinant viral vectors, such as those based on lentiviruses and adeno-associated viruses (AAVs), can confer long-term transgene expression in the host after a single dose. Particularly, recombinant (r)AAVs have emerged as favorable vectors, given their high in vivo transduction efficiency, proven clinical efficacy, and low immunogenicity profiles. Hence, rAAVs are being explored to deliver recombinant antibodies to confer immunity against infections or to diminish the severity of disease. When used as a vaccination vector for the delivery of antigens, rAAVs enable de novo synthesis of foreign proteins with the conformation and topology that resemble those of natural pathogens. However, technical hurdles like pre-existing immunity to the rAAV capsid and production of anti-drug antibodies can reduce the efficacy of rAAV-vectored immunotherapies. This review summarizes rAAV-based prophylactic and therapeutic strategies developed against infectious diseases that are currently being tested in pre-clinical and clinical studies. Technical challenges and potential solutions will also be discussed.

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

Abstract MP165: Exosome-mediated Encapsulation Alters AAV Antigenicity and Infectivity: Implications for Gene Delivery in the Heart

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Marta Adamiak ◽  
Yaxuan Liang ◽  
Cherrie Sherman ◽  
Shweta Lodha ◽  
Erik Kohlbrenner ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Cardiac Function ◽  
Neutralizing Antibodies ◽  
Firefly Luciferase ◽  
Interferometric Imaging ◽  
Human Cardiomyocytes ◽  
Clinical Benefits

Gene therapy is a promising approach for the treatment of cardiovascular disease. Current strategies for myocardial gene transfer include the use of adeno-associated virus (AAV) vectors. However, AAVs may not be ideal for gene therapy vectors owing to pre-existing AAV capsid immunity in the human population that may reduce transduction efficacy and hinder preclinical-to-clinical translation. Interestingly, recent studies suggest that exosome-mediated encapsulation may protect viruses from neutralizing antibodies (NAbs) against the capsid and promote viral infectivity. Here, we describe the ability of exosome-enveloped AAVs, i.e. exosomal AAVs (eAAVs), to evade NAbs and serve as a highly efficient gene delivery tool for cardiovascular therapeutics. We have developed a method to purifiy eAAVs from AAV-producing HEK-293T cells, and used electron/confocal microscopy, qPCR, immunoblotting, dynamic light scattering and interferometric imaging measurements to characterize eAAV morphology, contents and mechanism of action. We confirmed eAAVs represent vesicular fractions that exhibit common exosome phenotype, along with the presence of virus particles, and demonstrated that eAAV infectious entry potentially involves trafficking via endocytic compartments. Using flow cytometry, Langendorff perfusion system and bioluminescence imaging, we then evaluated efficiency of heart targeting for eAAV9/eAAV6 and standard AAV9/AAV6 encoding for mCherry or firefly luciferase in human cardiomyocytes in vitro and in mouse model in vivo . Regardless of the presence or absence of NAbs, we showed that eAAVs are more efficient in transduction in the same titer ranges as compared to standard AAVs. To test therapeutic efficacy, we intramyocardially injected eAAV9 or AAV9 vectors encoding for SERCA2a in NAb+ post-myocardial infarction mice and further evaluated cardiac function using echocardiography. Remarkably, eAAV9-SERCA2a outperformed standard AAVs significantly improving cardiac function in the presence of NAbs (%EF 55.14 ± 3.50 compared to 27.31 ± 1.63 at 6 weeks, respectively). In summary, delivery of AAVs protected by carrier exosomes (i.e. eAAVs) may retain the clinical benefits of AAVs while addressing one of its major challenges.

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