Why Are Lentivirus Vector Safety Tests So Crucial?

Replication Competent Lentivirus testing ensures safe administration of Cell and Gene Therapy products

Cell and gene therapies (CGT), also known as advanced therapy medicinal products (ATMPs) in Europe, have been the subject of extensive research for the treatment of genetic diseases and cancers. According to Informa Pharma Intelligence and the American Society of Gene and Cell Therapy ( Gene, Cell, & RNA Landscape, 2022 ), there are about 3,579 different gene, cell, and RNA therapies in preclinical development, with several already approved for treatment by regulatory agencies around the world.

Recent advances have included the use of carefully constructed, laboratory-generated virus particles that have been engineered for safety and efficacy. Virus genes are removed and replaced by non-viral genetic elements that can be used to treat patients. These particles are called viral vectors and have been generated based on several different virus types, each with their own advantages and disadvantages. One specific type is the lentivirus vector (LV), which can be used in gene and cell therapies.

LVs have been based on a modified version of the HIV virus. LVs gained popularity as a tool for stable gene transfer due to their large cloning capacity and their ability to efficiently transduce a broad range of dividing and non-diving host cells. The LV can enter a human cell and integrates its genetic element into the host cell genomic DNA providing stable long-term expression of desired functional genes to treat disease or fight cancer. LVs do not possess the ability to replicate and hence do not generate more virus particles. This state is called replication incompetence and makes the LVs safer to use as it limits the number of integration events into the patient cellular genome thus reducing the possibility of unwanted gene inactivation. The lack of replication also prevents additional cells being altered.

Rare recombination events within the plasmid sequences used to generate the LVs pose a risk of creating a Replication Competent Lentivirus (RCL). The presence of RCL events in vector batches increases the risk of insertional mutagenesis due to potential ongoing viral replication and multiple integrations into patient’s cellular DNA.

Of course, this lack of RCL in preparations of LVs needs to be tested thoroughly to ensure safety. A recent US Food and Drug Administration (FDA) guidance for Industry, Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-up (JAN 2020) , suggests that RCL testing should be performed on the vector producer cells, the end-of-production cells, the vector stock in the cell culture supernatant, and in the LV-modified patient cells. Current recommendations include testing samples from multiple stages of the manufacturing processes. The FDA, European Medicines Agency (EMA), and most other regulatory agencies require extensive testing for RCL in retroviral vector products as well as in patients as a long-term follow-up.

Due to potential risks of the procedure, the regulatory authorities have mandated testing the production batches of the LVs as well as the cells used in their manufacturing. This is a must-do release test and therefore the test must be designed to be robust and sensitive enough to catch even a single RCL event that may occur in each patient dose. In conclusion, LVs and cell therapy products modified using LVs need to be thoroughly investigated before being administered to patients and tests like these go a long way to ensure patient safety.

Testing for RCL at Charles River Laboratories involves passaging potential virus material in cell culture multiple times, using a permissive cell line like C8166 in the amplification phase. Subsequently, the amplification phase material is further passaged in an additional indicator phase to capture any low-level RCL event. If an RCL is present, it will generate virus particles during growth in the cell culture and these virus particles would be found in the cell-culture supernatant. Cell culture supernatants harvested from these passages are subjected to two different end-point assays that conclusively prove the presence of a live and replicating lentiviruses. One of these assays involves detection of an enzyme found in a retrovirus, reverse transcriptase; while the other test is used to quantitate the level of the LV capsid protein, p24. When one or both these tests are positive, they prove the presence of a live replicating virus in the LV manufactured batch.

From blood cancers to solid tumor, LVs have great promise in giving patients hopes and cures from hard-to-treat cancers and other debilitating diseases. We expect to see more treatments come available in addition to those currently approved by FDA. Hope in action.

Viral Safety Testing

Charles River offers a portfolio of in vivo, in vitro, and biochemical viral safety assays, which can be customized to address the specific needs of a client.

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Charles River Laboratories has developed a battery of vector-specific assays, including but not limited to replication competent assays, for characterization and safety testing.

Altaf Kazi, PhD, is Associate Director, Methods Development for Charles River's PA Biologics site.