ADA Assays Screening Viral Vectors For Gene Therapy
By integrating modified or new genes into the cells of the human body, gene therapy has the potential to treat a wide variety of acquired and inherited diseases. Genetic therapeutics must deliver the intended genetic material to a large quantity of specific cells within the correct tissue. Properly targeting cells requires that the genetic material reaches the intended targets without delivering the material to the wrong cells.
By copying a viral capsid to transport the gene, researchers can better predict how the therapeutic will bind with target cells. However, pre-existing anti-drug antibodies (ADAs) will view the viral capsid containing the gene, as any other virus- interfering with the intended pharmacokinetic process.
Types of Viral Vectors for Gene Therapy
Viruses have naturally evolved to find and enter specific human cells for the purpose of replicating their genetic material. By replacing the viral genes with therapeutic genes, the treatment uses the vectors’ delivery method. There are four types of viral vectors, each with differing characteristics and applications, adeno-associated viral, adenoviral, lentiviral, and retroviral.
AAV vectors, or adeno-associated viral vectors, are non-enveloped single strand paroviruses. They are limited in that they can only deliver smaller amounts of DNA. These vectors are ideal for in vivo therapy, which means they are delivered to targeted cells within the patient’s body. In contrast, ex vivio treatment involves removing targeted cells from the body, treating them with vecotrs, and then returning the modified cells to the body
Usually, AAV treatment does not become a part of the cell’s genome, meaning that a dividing cell will lose the treatment’s effect over time. Therefore, AAV is better for non-dividing cells such as those in the nervous system or liver. Within these cells, AAVs can last for many years– or even a person’s lifetime.
Adenoviral vectors are non-enveloped like AAVs, but they are double-stranded and can deliver almost eight times as much genetic material. The nonintegrating delivery method leads to lost expression after cell division. While early adenoviral vectors were easy to produce and were commonly used in research, they induce a pronounced inflammatory immune response that slowed clinical applications.
Lentiviral and Retroviral Vectors
These vectors can transport larger amounts of RNA, which then becomes DNA. These vectors are unique in that their genetic information is integrated into the target cell, allowing modifications to ensure throughout the cell’s replication. Because of this, lentiviral and retroviral vectors are preferred for dividing cells, and the dividing cells are injected ex vivo, or outside of the body.
Immune Responses to Viral Gene Therapy Vectors
Immunogenicity is the ability of a substance to provoke an immune response. As is clear from the above, a key hurdle to viral therapy is the patient’s immune response. An immue response can render the therapy ineffective, or worse, cause a life-threatening reaction such as anaphylaxis. Unfortunately, immunogenicity will remain a considerable barrier to gene therapies. In comparison, non-viral vectors for gene therapy are less likely to ignite an immune response, and they are less expensive to manufacture. However, current non-viral vector experience is limited. Because the immune response is an essential part of viral vector treatment, immunogenicity testing is crucial.
Some Viral Vector Immunogenicity Data
A problem with viral vectors is that they have a high or quickly produce immunogenicity in a patient: as an example, 30-70% of patients have pre-existing antibodies to AAVs, depending on the exact type in question. This is because many people have been exposed to them in past infections, and their immune systems learned to attack them. In these cases, the patient’s antibodies destroy the therapeutic vessel before it can deliver the gene, making treatment difficult.
At the same time, patients may develop antibodies after just one treatment, so it can also be limited in this way. Scientists are working to combat this problem with artificial vessel exteriors and more.
Pre-Screening for ADAs and Their Neutralizing Affect Before Adding Gene
ADA, or anti-drug antibody, analysis determines the ADA response—a necessary step to ensure safety and effectiveness for any novel therapeutic drug. The first assay is a bridging assay, which approximates the number of ADAs. For this, an anti-AAV antibody assays on the MSD (meso scale discovery assay) is superior to the ELISA.
MSD ADA assays, based on electrochemiluminescence, are highly sensitive, have a big dynamic range, and have good flexibility. The format provides more data with less material, allowing for the detection of multiple proteins in a single sample. MSD assays detect low-affinity ADAs to the viral vectors.
When ADAs are observed in patients, Nab (neutralizing antibody) assays are often necessary in a clinical trial. If the present ADAs neutralize the therapeutic drug, the drug’s effectiveness could be altered. Using cell-based Nab assays, researchers model the way Nabs work in vivo. While valuable in observing Nabs, cell-based screening of ADAs can neutralize the effect of the viral vector to bind and enter the observed cells. These assays rely on an effective structure, which requires a deep understanding of how the vectors bind to the host cell – making these tests difficult to execute.
Regulatory Requirements for Developing and Validating ADA Assays
The FDA recommends a multi-tiered testing approach, consisting of a screening assay, a confirmatory assay, and a titration and neutralization assay. In this study design, the screening assay is used to detect antibodies, the confirmatory assay to confirm that the antibodies are in fact specifically bonding to the therapeutic drug, and the titration and neutralization assays to analyze the magnitude of the immunogenic response. In addition, titration and neutralization assays determine if the antibody is demonstrating neutralizing activity. For neutralizing antibody analysis, the FDA recommends at least 30 samples.
For the viral vector screening assay, all relevant immunoglobulin (Ig) isotypes should be detected. When there is no anaphylaxis risk and the administration route is not mucosal, the isotypes are IgM and IgG. For mucosal administration routes, IgA isotypes are also important.
Therefore pre-screening of viral vectors and thus modification of the vector in gene therapy could provide promising treatment opportunities. If you’re looking for an expert team with knowledge of gene therapy vectors to develop ADA and Nab assays, Sword Bio can help. Connect with one of our scientists to discuss your project.