How to Succeed with Gene Therapy Applications When Experimental Reproducibility is Critical
Introduction
Gene therapies hold great promise for the delivery of transformative technologies in the coming years. These targeted, complex therapies, which usually work by introducing vectors containing genes into a patient, have the potential to cure rare and difficult-to-treat diseases across a range of medical areas. However, several challenges remain to be addressed before gene therapy can deliver on its promise. One of the most important is experimental reproducibility in the research and discovery stages, which usually take place in an academic setting. Despite the great importance of reproducibility, it is not uncommon to hear of fellow scientists trying and failing to reproduce someone else's results.
"The success of gene therapy is reliant on the development of reproducible data."
There are endless scientific approaches and complex technical procedures involved in a process and, consequently, an experimental result can be challenging to replicate. All parties involved in the development and delivery of next-generation medicines need to rely on robust, data-derived evidence of their benefits in order to support decision making and to ensure translation from promising discoveries to effective therapeutics.
Here, we highlight some obstacles to achieving experimental reproducibility that are associated with lab equipment and provide recommendations on how they can be overcome. Achieving the highest level of uniformity among common lab practices is an absolute must. This means lab scientists should be sufficiently trained and should follow strictly the correct procedures for operating and maintaining lab equipment. In academic research settings, these protocols are design by lab managers based on recommendations found in the user manuals provided by equipment suppliers. Since there are no specific rules to follow, academic practices can vary dramatically. Thermo Fisher Scientific recognizes the need to provide additional support in the form of a variety of comprehensive materials, including technical, application, and smart notes, covering every aspect of equipment use and maintenance. All with the aim of allowing you to make the most of your equipment and to increase the reproducibility of your experimental data.
Improving Research-Scale Laboratory Practices
Plasmid Production
In an academic setting, plasmid encoding viral vectors are usually produced in Escherichia coli bacterial cells on orbital shakers such as the Thermo Scientific™ Solaris™ Benchtop Temperature Controlled Shakers. If the aim is to achieve high quality plasmid, in quantity and in a reproducible manner, some key features need to be taken into consideration. For example, shaker speeds of up to 400rpm have been shown to produce higher yields and limiting the circulation of microorganisms, which could potentially contaminate the culture, is important for ensuring the integrity of the final product. Similarly, features which facilitate regular, easy cleaning ensure that no surfaces harbor residual contaminants. Furthermore, the ability to simultaneously view and modify parameters while the shaker is in operation are beneficial in ensuring quality control. A smart note1 dedicated to important considerations when evaluating a large orbital shaker for production of plasmid DNA is available.
Suspensions need to be agitated at different speeds and with different orbits depending on the culture vessel and cell type, hence choosing the right orbital shaker for your application is critical.2 Other elements, like correct shaker installation, operation and cell production best practice, cleaning and disinfection processes, and regular preventive maintenance are also critical to increasing experimental reproducibility.3 Thermo Fisher Scientific is always a step ahead and has invested a lot of time and resources in order to optimize the most needed protocols (e.g. plasmid DNA production in E. coli4), so you don’t need to. You can increase efficiency and at the same time increase experimental reproducibility by testing and potentially implementing these protocols in your lab.
Other critical activities in plasmid DNA preparation are bacterial cell pelleting and plasmid extraction and purification. In an academic setting, centrifugation is still the most popular and cost effective way to execute these steps. For instance, plasmid DNA can be isolated in a cesium chloride gradient, but this method requires an ultracentrifuge such as the Thermo Scientific™ Sorvall™ WX+ and utilizes ethidium bromide. Alternatively, gravity or spin column kits can effectively purify plasmid DNA in superspeed and microcentrifuges. To increase the reproducibility of your protocol a Thermo Scientific™ Sorvall™ LYNX 6000 Superspeed Centrifuge and Thermo Scientific™ Fiberlite Carbon Fiber Rotors can be used in concert with commercially available DNA preparation kits to obtain high quality plasmid DNA on a large-scale (about 1mg). Alternatively, a lower cost method is available without using a kit. Both protocols have been optimized and recommendations are easily accessible in the form of an application note.5 In addition, Thermo Fisher Scientific provide comprehensive user manuals for all their centrifuge models, such as for the LYNX,6 which include disinfection, decontamination, autoclaving, and maintenance recommendations that can be effortlessly translated into step-by-step operating procedures. Thus you can not only increase the life of a centrifuge but also increase the success rate and reproducibility of your plasmid extraction procedures. Furthermore, if you are planning to move from research operations into a GLP/GMP-regulated environment in the future, then Thermo Scientific™ Centri-Log™ Plus Data Management Software7 can be incorporated into Sorvall™ LYNX centrifuges in order to enable accurate data collection and traceability in compliance with 21 CFR Part 11 rules for electronic data recording.
Viral Vector Production
Developing and optimizing your lentiviral production platform in order to create robust transfection protocols can become very tricky, especially when high-yield, scalability, and reproducibility are your priorities. All components, including media, transfection reagents, supplements, and cells, need to work in perfect synergy to generate superior and functional lentiviral particles. Research applications, such as creating new gene therapies, depend hugely on high-quality cell growth. An illustrative example of this is in the use of an adhesive or suspension culture for HEK 293 viral production cells. Here, if the cells are not grown under optimum conditions, both experimental readout and reproducibility will be negatively affected in ways difficult to detect.
The quality attributes of the final cell product, viral particles, can vary greatly depending on the culture conditions sustained in the CO2 incubator. In fact, reproducible culturing throughput can be greatly affected by implementing different features and technologies in CO2 incubator design. As cells respond to changing or different cues, it is crucial that the conditions in the culturing chamber are uniform from top to bottom and side to side, such that all cells experience the same conditions. It is even more important that the incubator recovers quickly to desired conditions following door opening, so that cells spend their maximum time at their ideal parameters, speeding doubling time as well as helping to ensure quality. Ideal conditions for cell health and cell growth are not just related to temperature, as CO2 level, oxygen level, and relative humidity (RH) are also important, as all play a role in cell health. Incubators that ensure uniformity and fast recovery, like the Thermo Scientific™ Heracell™ VIOS CO2 and Forma™ Steri-Cycle™ CO2 incubators, will help to increase lot-to-lot reproducibility. In addition, Thermo Fisher Scientific provides best practice information for proper care and maintenance of your cell culture incubator, including advice on installation and positioning in the lab, reducing contamination, and disinfection procedures, as well as recommendations for using the correct type of water. All with the aim of helping to ensure quality experimental results that will drive your discovery.8,9
If you are growing your HEK 293 lentiviral production cells in suspension, you may benefit from using a small footprint CO2-resistant orbital shaker. Ideal for use in a CO2 incubator, these shakers have specially-treated mechanical components designed to withstand up to 20% CO2 and 95% humidity. By comparison, most open-air orbital shakers are not designed to withstand the acidic conditions of a CO2 chamber and must be replaced on a regular basis. Thermo Scientific™ CO2-Resistant Orbital Shakers are also designed to dissipate minimal heat, so as to ensure that conditions within the CO2 incubator chamber are not affected. The correct choice of orbital shaker for your application can extend its life as well as make sure that optimal growth conditions and reproducibility in culturing results are maintained.
Conclusions
There are many examples of laboratory equipment present in gene therapy research that you may not think about but which have significant potential to impact the reproducibility of your experimental results. The critical lesson to keep in mind is to pay more attention and dedicate more time to familiarizing yourself with supplier recommendations, as provided in user manuals and varied technical notes. In experimental research settings, it is common to focus mainly on specific experimental protocols, dismissing specific recommendations for equipment installation and proper positioning, as well as care and preventative maintenance, contamination prevention, and cleaning and disinfection. Remember that even lab equipment like biosafety cabinets, centrifuges, incubators, shakers, fridges, and freezers need to be treated with respect and a lot of care. They need to become a partner in your journey towards making your experiments both more successful and more reproducible.
References
1 Thermo Scientific. Smart Note: Orbital Shakers-Which Features are Important Considerations When Evaluating a Large Orbital Shaker for Production of Plasmid DNA Encoding Viral Vectors, for Applications Including CAR-T, Gene Therapy, or Other Genetic Engineering? https://assets.thermofisher.com/TFS-Assets/LPD/Product-Information/Orbital-Shakers-DNA-Encoding-SmartNote-SNORBSHAKERDNA-EN.pdf
2 Thermo Scientific. Application Note: Choosing the Right Orbital Shaker for Your Application. https://assets.thermofisher.com/TFS-Assets/LED/Application-Notes/D20064.pdf
3 Thermo Scientific. Application Note: Orbital Shaker Benchmarks-Best Practices for Use and Maintenance. https://assets.thermofisher.com/TFS-Assets/LED/Application-Notes/Orbital-Shaker-Benchmarks-Best-Practices-App-Note-ANMAXQBEST.pdf
4 Thermo Scientific. Application Note: Optimization of Plasmid DNA Production in Escherichia coli Utilizing the Thermo Scientific MaxQ 8000 Incubated Stackable Shakers. https://assets.thermofisher.com/TFS-Assets/LED/Application-Notes/D19993~.pdf
5 Thermo Scientific. Application Note: DNA Preparation Using the Thermo Scientific Sorvall LYNX Superspeed Centrifuge Series. https://assets.thermofisher.com/TFS-Assets/LED/Application-Notes/D21231.pdf
6 Thermo Scientific. Instruction Manual: Thermo Scientific Sorvall LYNX 4000/6000 Superspeed Centrifuge. https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLED%2Fmanuals%2F50136519-h-Thermo%20Scientific%20Sorvall%20LYNX-en.pdf
7 Thermo Scientific. Centri-Log Plus Software: An Accurate and Reliable Data Management Solution. https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLPD%2Fbrochures%2FCentriLog-Plus-Brochure-GLOBAL-FNL-FWR-1.pdf
8 Thermo Scientific. Technical Note: Proper Care and Maintenance for a Cell Culture Incubator. https://assets.thermofisher.com/TFS-Assets/LED/Warranties/TNCO2CAREFEED-EN.pdf
9 Thermo Scientific. Smart Note: CO2 Incubators-Smart Water for Your CO2 Incubator. https://assets.thermofisher.com/TFS-Assets/LED/brochures/CO2-Incubator-Water-SmartNote-EN.pdf
