Gene Therapy

Gene therapy, which delivers DNA into a patient’s cells to replace or add genes that may be abnormal or missing, is starting to deliver results in the clinic after 30 years of development. With the FDA approval of the second adeno-associated virus (AAV) based gene therapy treatment in May of 20191, it is truly an exciting time in the field. AAV has proven to be a powerful tool for gene therapy purposes for its broad tropism, inability to replicate on its own in vivo, minimal immunogenicity, and ability to deliver effective and long-lasting results2. However, there are several challenges related to the development and manufacturing of AAV-based processes, including characterization, quantification, and downstream purification3, 4.

What Are The Challenges of Manufacturing Gene Therapies?

Manufacturing challenges in particular include the need for the consistent production of high purity, high potency, and robust safety for AAV products, all while maintaining acceptable large-scale manufacturing costs5. While there are several methods for AAV production, including transient transfection in human (HEK293) cells6, baculovirus driven production in insect (Sf9) cells7, or recombinant helper viruses such as HSV in mammalian cells8, 9, the importance of precise and predictive analytics remains regardless of the production system. LumaCyte’s Radiance® instrument, which uses Laser Force CytologyTM (LFC) to measure the intrinsic biophysical and biochemical properties of single cells10, 11, has the potential to significantly improve the characterization of cell-based AAV transfection and production, improving the efficiency and accuracy of both processes and shortening development time

Some key gene therapy applications for Radiance® Laser Force Cytology™ include:

Rapid detection and quantification of cellular properties during viral vector production either via plasmid transfection (AAV, lentivirus) or viral infection (baculovirus, adenovirus, or HSV), including titer and packaging efficiency. In process results provided by Radiance® can be correlated to existing assays such as ddPCR, ELISA, or TCID50 to provide results in just minutes. This improves the speed and quality of process development by reducing time to results and the need to run tedious and labor-intensive assays that require significant time and resources.

When used as a process analytical technology (PAT) during production/manufacturing, Radiance® can provide rapid information on how input cell and virus raw materials vary over time, and then real-time analytics to help scientists understand performance and variation throughout a production run, thereby increasing process knowledge to ensure consistent performance and reduce the occurrence of out of spec batches. Radiance® is GMP ready and designed for ease of use by production personnel. Both the Illuminate software that powers Radiance® and LumaCyte’s cloud-based analytics platform ReportR are 21 CFR Part 11 compliant.

For analytical development, Radiance® provides viral transduction, infectivity and other cell-based assays for gene therapy R&D, process development and optimization, formulations, and potency.  Precise viral analytics allow for more accurate vial filling to prevent the need for over formulation and provide a better prediction of shelf life.
Radiance® enables label-free viral neutralization assays with high accuracy and precision as well as a large dynamic range for clinical trial and other applications. This facilitates the rapid screening of patients for previous exposure to viral vectors as well as the evaluation of immunological response as a result of treatment.

AAV Production

Transfection

Transfection in gene therapy is generally used for the production of the viral vector intended to deliver the gene of interest into the patient. Typically, this process begins with the insertion of plasmids into the production cells via chemical means of opening the membrane temporarily to insert the plasmids. Chemical transfection is the most common method as it is simple and inexpensive to use compared with more sophisticated methods such as electroporation or microinjection. Most AAV therapies use chemical transfection in human embryonic kidney HEK 293 cells for the production of the indented AAV virus. Selecting the optimal transfection reagent and conditions is an important step for R&D and process development to ensure and efficient production process. As one example, Radiance has been used in many different gene therapy situations to quickly identify high efficiency transfection reagents. The results correlate well with ddPCR results, as shown in the tech note below and can be delivered in near-real time throughout a process.

Label-Free Monitoring of AAV Transfection in HEK293 Cells Using Laser Force Cytology™

LumaCyte’s label-free LFC technology yields a wealth of data, delivering deep insights into cellular conditions which are incredibly valuable in the monitoring of production quality, including cell health and potential contamination, in addition to viral titer.