CÔNG TY TNHH THIẾT BỊ KHOA HỌC KỸ THUẬT AN DƯƠNG
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To treat genetic diseases using gene therapy, recombinant adeno-associated viruses (AAVs) are under
active investigation as vectors for gene delivery. When used for gene delivery, an AAV can achieve
long-term, stable transient gene expression. An important advantage of AAVs as gene therapy vectors,
compared to alternative approaches, is the ability to target gene delivery to specific tissues or cell types
with cell type–specific AAV variants.
Để điều trị các bệnh di truyền bằng liệu pháp gen, các virus tái kết hợp adeno-associated (AAVs) đang được nghiên cứu tích cực như các vectơ cho việc giao tiếp gen. Khi sử dụng cho việc giao tiếp gen, một AAV có thể đạt được sự biểu hiện gen tạm thời ổn định lâu dài. Một ưu điểm quan trọng của AAVs như các vectơ điều trị gen, so với các phương pháp thay thế, là khả năng nhằm mục tiêu giao tiếp gen đến các mô hoặc loại tế bào cụ thể bằng các biến thể AAV theo loại tế bào.
In recent work published in the Journal of Biological Engineering, Chen et al., describe a new biomanufacturing
platform to produce AAV therapeutics that has been developed to be robust and scalable. Typically, AAV is
produced in cultured cells, where the yield depends on both the amount of AAV produced by the cells and the percent of produced virus that can be recovered and purified from the culture. The authors state that current platforms are insufficient to meet demands for AAV productivity and rate of recovery. The researchers used a Cielo qPCR System and the Azure 300 Imaging System in the course of validating a new platform designed to optimize productivity and recovery (Figure 6).
To develop the new biomanufacturing platform, the group evaluated and optimized each step of the AAV production and purification process, including selection of the host cell type, transfection parameters, culture clarification, column purification, and concentration and storage of the virus. In addition, the morphology, activity, and gene expression of the manufactured AAVs were characterized. The authors note the platform is applicable to multiple AAV serotypes.
The new platform involves producing AAV in transfected cells grown in liquid culture. The authors found VPC cells produced 5-times more AAV than HEK cells. They identified the optimal cell density and plasmid DNA to cell ratio for optimal transfection, and validated these conditions with four AAV serotypes. Fluorescent qPCR with SYBR Green detection on the Cielo was used to quantify the amount of AAV in culture. The authors assessed virus production over time, finding the optimal collection point and identified conditions to achieve 95%-100% AAV release efficiency. To scale up production, they moved production to stirred-tank bioreactors and again characterized cell growth and AAV titer over time.
Producing sufficient AAV for clinical applications requires more than maximizing the amount of AAV produced by cultured cells. It is also important to extract and purify as much of the AAV as possible, while maintaining the activity of the virus. The authors characterized multiple methods of column purification to identify the combination of column, buffer, and elution profiles with the highest binding and elution rates. SDS PAGE and Western blotting were used to assess the quality of the AAV produced. Silver-stained protein gels and chemiluminescent Western blots were both imaged on the Azure 300 to detect AAV capsid proteins (see attached figure). The authors demonstrated the AAV produced in their system was active and drove functional gene expression in cell culture and in mice.
The authors conclude that the reported biomanufacturing platform is robust, scalable, and results in a high recovery rate of high-quality AAV. They emphasize that the process can be used with different AAV serotypes and could benefit numerous research and clinical applications.
In this study, the authors took advantage of the Azure 300 Imaging System’s ability to carry out white light (silver stain) and chemiluminescence imaging and utilized a Cielo qPCR System to quantify AAV with SYBR Green.
The Azure 300 can conduct UV and blue-light imaging and is capable of being fully upgraded to an Azure 600, a fluorescent Western blot imaging system. Learn more about the Azure 300 here.
The Azure 300 offers the simplicity, speed and sensitivity of film detection, with better resolution and more quantitative results. This upgradeable chemiluminescent imager replaces a darkroom with film, while providing accurate and fast chemiluminescent detection, as well as the sensitivity, dynamic range, and linearity needed for quantitative blot analysis.
Viral titers were assessed using a Cielo. This robust qPCR system incorporates individual well scanning with channel-specific excitation and emissions fiber optics at each well for increased sensitivity and signal-to-noise. Learn more about the Cielo here.
Hundreds of clinical trials are presently studying the use of AAV-delivered gene therapies to treat numerous diseases. For any successful therapy to be produced and provided to patients, it will be necessary that the AAV therapeutic be manufactured at a large scale while maintaining activity and quality.
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