Lentivirus is a widely used viral vector in gene therapy. It offers several advantages over other viral and non-viral vectors, such as the ability to carry large or multiple genes into target cells, infect both dividing and non-dividing cells, and integrate transgenes directly into the host cell’s genome.
However, these desirable Lentiviral vector properties require a more complex structure compared to other common viral vectors, such as the adeno-associated virus (AAV). Lentivirus is an enveloped, spherical virus, with a diameter between 90-130 nm, and consists of multiple components, including a transgene, nucleocapsid, capsid, envelope, and surface membrane proteins.
The complex structure of lentivirus presents analytical challenges when measuring attributes like size and titer, which are essential for optimizing its stability, efficacy, and storage conditions.
Performing Lentiviral transfection and functional tittering assays after different storage conditions can produce wildly different results, indicating that colloidal and morphological changes to the lentivirus structure have a significant impact on its efficacy as a gene therapy vector.
In this application note, we present Zetasizer Advance Ultra and NanoSight Pro thermal stability data for size, polydispersity, and viral particle titer. Each technology performed measurements over a thermal ramp from low to high temperatures.
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Lentivirus is a widely used viral vector in gene therapy. It offers several advantages over other viral and non-viral vectors, such as the ability to carry large or multiple genes into target cells, infect both dividing and non-dividing cells, and integrate transgenes directly into the host cell’s genome1.
However, these desirable Lentiviral vector properties require a more complex structure compared to other common viral vectors, such as the adeno-associated virus (AAV). Lentivirus is an enveloped, spherical virus, with a diameter between 90-130 nm, and consists of multiple components, including a transgene, nucleocapsid, capsid, envelope, and surface membrane proteins2.
The complex structure of lentivirus presents analytical challenges when measuring attributes like size and titer, which are essential for optimizing its stability, efficacy, and storage conditions.
Performing Lentiviral transfection and functional tittering assays after different storage conditions can produce wildly different results, indicating that colloidal and morphological changes to the lentivirus structure have a significant impact on its efficacy as a gene therapy vector.
In this application note, we present Zetasizer Advance Ultra and NanoSight Pro thermal stability data for size, polydispersity, and viral particle titer. Each technology performed measurements over a thermal ramp from low to high temperatures.
NanoSight Pro utilizes Nanoparticle Tracking Analysis (NTA)3 to measure nanoparticle size and concentration. Particle size is determined by quantifying the mean squared displacement of multiple individual particles. Particle concentration, or viral titer, is calculated by counting the number of viral particles in a known volume. NTA size and concentration measurements occur simultaneously and require about five minutes per sample.
The Zetasizer Advance Ultra Red uses dynamic light scattering (DLS)4 to measure particle size and multi-angle dynamic light scattering (MADLS)5 to measure high resolution size and particle concentration6. Particle concentration is calculated from the measured MADLS size, the material scatter rate, material refractive index, dispersant viscosity and refractive index.
Ultra-purified lentivirus samples were purchased from Vector Builder produced with the pLV[Exp]-EGFP:T2A:Puro-EF1A>mCherry vector.
Zetasizer Advance Ultra size and concentration measurements were performed on neat lentivirus samples by pipetting 20 microliters of freshly thawed sample directly into the ZEN2112 quartz cuvette inside of a BSL-2 hood. DLS and MADLS measurements were performed at 5-degree intervals from 20 to 100oC. All sample handling was performed inside a BSL-2 hood, and bleach was used to disinfect all cuvettes at end of the experiment.
NanoSight Pro measurements were performed by diluting samples 1000x into HBSS buffer immediately prior to analysis using the NanoSight Pro using NS Xplorer software version 1.1. Samples were measured using autofocus and camera setup algorithms. All sample preparation and measurements were performed inside a BSL-2 hood, including the operation of the NanoSight Pro instrument. NTA measurements were performed at 5-degree intervals from 25oC to 70oC. The flow cell was disinfected by flushing with 20% bleach followed by water.
Both the Zetasizer Advance Ultra and NanoSight Pro measured a size change at 60oC (Figure 1 and 2). This indicates that the lentivirus sample lost colloidal stability at this temperature, resulting in an increase to the measured size, likely caused by the formation of aggregates. Zetasizer Advance Ultra saw the intensity-weighted average size increase to over 300 nm before stabilizing at 320 nm from 80 to 95 degrees. The NanoSight Pro number-weighted size measured a change in mode size from 130 nm to 219 nm, suggesting the size change was due to a loss of lentivirus monomers in the sample.
![[Figure 1 v2 an240913-lentivirus-thermal-stability.jpg] Figure 1 v2 an240913-lentivirus-thermal-stability.jpg](https://dam.malvernpanalytical.com/3985da0d-4f9e-4dc2-b2c2-b20d00f28421/Figure%201%20v2%20an240913-lentivirus-thermal-stability_Original%20file.jpg)
Figure 1. Zetasizer Advance Ultra size results for lentivirus heated from 20-100°C (arrow indicates thermal aggregation point).
![[Figure 2 v2 an240913-lentivirus-thermal-stability.jpg] Figure 2 v2 an240913-lentivirus-thermal-stability.jpg](https://dam.malvernpanalytical.com/3dd4f7b3-b2c9-4ca8-8933-b20d00f28411/Figure%202%20v2%20an240913-lentivirus-thermal-stability_Original%20file.jpg)
Figure 2. NanoSight Pro size results for lentivirus heated from 25-70°C (arrow indicates thermal aggregation point).
Both technologies also measured a reduction in total lentivirus particle concentration over the thermal ramp. The Zetasizer Advance Ultra measured the largest drop in lentivirus particle concentration at 60°C, coinciding with the change in size. This is unsurprising since the Zetasizer Advance Ultra calculates particle concentration partly from the measured size produced by the MADLS size distribution. The NanoSight Pro observed a more steady reduction in lentivirus particle concentration starting from 40 degrees to 70°C. These results highlight the difference between particle concentration calculated from a native intensity distribution compared to a number distribution.
![[Figure 3 AN240913-lentivirus-thermal-stability.jpg] Figure 3 AN240913-lentivirus-thermal-stability.jpg](https://dam.malvernpanalytical.com/2ca84062-c6c6-4892-8207-b20900dd37ad/Figure%203%20AN240913-lentivirus-thermal-stability_Original%20file.jpg)
Figure 3. Zetasizer Advance Ultra and NanoSight Pro lentivirus titer particle concentration thermal ramp results.
Both the Zetasizer Advance Ultra and NanoSight Pro were successfully able to measure lentivirus over a temperature range. The particle sizing technologies observed a significant change to colloidal stability, measured as a change in size, at 60°C.
Thermal stability can predict the long-term stability of pharmaceuticals, which can impact safety and efficacy. By improving lentivirus structure and liquid formulations, the efficacy and stability of future lentivirus-based gene therapies can be improved. The results stress the importance of performing both vector nanoparticle characterization using multiple, orthogonal technologies in addition to functional infectious assays.
| Product name | View online |
|---|---|
| Zetasizer Advance Ultra | |
| NanoSight Pro |
