Confirmatory Detection of Neutralizing Antibodies to AAV Gene Therapy Using a Cell-based Transduction Inhibition Assay

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.

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Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout

Virology Journal ◽

10.1186/s12985-021-01501-7 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Tugba Mehmetoglu-Gurbuz ◽

Rose Yeh ◽

Himanshu Garg ◽

Anjali Joshi

Keyword(s):

Gene Therapy ◽

Lentiviral Vector ◽

Suicide Gene ◽

Vector System ◽

Tat Protein ◽

Ccr5 Gene ◽

Hematopoietic Stem ◽

Therapy Strategy ◽

Hiv Tat ◽

A Cell

Abstract Background Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone. Methods A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein. Results Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir. Conclusions Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.

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Proceedings of the first academic symposium on developing, qualifying and operating a cell and gene therapy manufacturing facility

Cytotherapy ◽

10.1016/j.jcyt.2018.07.008 ◽

2018 ◽

Vol 20 (12) ◽

pp. 1486-1494 ◽

Cited By ~ 4

Author(s):

DAVID L. DIGIUSTO ◽

KATHRYN MELSOP ◽

RASHI SRIVASTAVA ◽

CHY-ANH T. TRAN

Keyword(s):

Gene Therapy ◽

Manufacturing Facility ◽

A Cell ◽

Cell And Gene Therapy

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Implementation of design of experiments (DOE) in the development and validation of a cell-based bioassay for the detection of anti-drug neutralizing antibodies in human serum

Journal of Immunological Methods ◽

10.1016/j.jim.2011.11.004 ◽

2012 ◽

Vol 376 (1-2) ◽

pp. 32-45 ◽

Cited By ~ 16

Author(s):

Xinyi C. Chen ◽

Lei Zhou ◽

Shalini Gupta ◽

Francesca Civoli

Keyword(s):

Design Of Experiments ◽

Human Serum ◽

Neutralizing Antibodies ◽

A Cell ◽

Development And Validation

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SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses

10.1101/2021.03.10.21253299 ◽

2021 ◽

Author(s):

Raymond T. Suhandynata ◽

Nicholas J. Bevins ◽

Jenny T. Tran ◽

Deli Huang ◽

Melissa A. Hoffman ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Adaptive Immune Response ◽

Antibody Assay ◽

Adaptive Immune Responses ◽

Adaptive Immune ◽

Previous Infection ◽

A Cell

AbstractBackgroundThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated.MethodsThe ability of four commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 SARS-CoV-2 PCR-positive patients, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 SARS-CoV-2 negative patients. Serology results were compared to a cell based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay.ResultsThe Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received two-doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme Nabs assay correlated with the PSV SARS-CoV-2 ID50 neutralization titers (R2= 0.70), while correlation of the Roche S-antibody assay was weaker (R2= 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received two-doses of the Moderna vaccine (ID50: 597) compared to individuals that received a single dose (ID50: 284).ConclusionsThe Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.SummaryThe Roche S (spike protein)-antibody and Diazyme neutralizing-antibodies (NAbs) assays were evaluated for their clinical utility in the detection of SARS-CoV-2 related adaptive immune responses by testing SARS-CoV-2 PCR-confirmed patients, SARS-CoV-2-vaccinated individuals, and SARS-CoV-2-negative individuals. Commercial serology results were compared to results generated using a cell-based SARS-CoV-2 pseudovirus (PSV) NAbs assay and previously validated SARS-CoV-2 commercial serology assays (Roche N (nucleocapsid protein) antibody and Diazyme IgG). We demonstrate that the Roche S-antibody and Diazyme NAbs assays detected adaptive immune response in SARS-CoV-2 vaccinated individuals and the presence of SARS-CoV-2 PSV NAbs. The Roche S-antibody assay had an observed positive percent agreement (PPA) of 100% for individuals who received two doses of the Pfizer or Moderna vaccine. By contrast, the Roche N assay and Diazyme IgG assay did not detect vaccine adaptive immune responses. Our findings also indicate that the Diazyme NAbs assay correlates strongly with the levels of SARS-CoV-2 ID50 neutralization titers using the PSV Nab assay in vaccinated individuals.

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

Circulation Research ◽

10.1161/res.127.suppl_1.mp165 ◽

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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Gene Therapy: The Molecular Bandage for Treating Genetic Disorders

Anwer Khan Modern Medical College Journal ◽

10.3329/akmmcj.v3i1.10110 ◽

1970 ◽

Vol 3 (1) ◽

pp. 24-27

Author(s):

Md Manjurul Karim

Keyword(s):

Gene Therapy ◽

Viral Vectors ◽

Genetic Disorders ◽

Clinical Status ◽

Genetic Material ◽

Review Article ◽

Cell Tissue ◽

Efficient Gene ◽

Effective Delivery ◽

A Cell

The concept of gene therapy involves the transfer of genetic material into a cell, tissue, or whole organ, with a view to curing a disease or at least improving the clinical status of a patient. Much of its success relies heavily on the development of an effective delivery system that is capable of efficient gene transfer in a variety of tissues, without causing any associated pathogenic effects. Viral vectors currently offer the best choice for efficient gene delivery, what has been discussed in this review article. Their performance and pathogenecity has been evaluated in animal models, and encouraging results form the basis for clinical trials to treat genetic disorders and acquired diseases. Despite some initial success in these trials, vector development remains a seminal concern for improved gene therapy technologies. DOI: http://dx.doi.org/10.3329/akmmcj.v3i1.10110 AKMMCJ 2012; 3(1): 24-27

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