Gene therapy is a therapeutic strategy based on the modification of gene expression within target cells via the employment of viral or non-viral vectors. Up to date, most of the US Food and Drug Administration (FDA)-approved gene therapies are viral based (Wang et al., 2024 Signal Transduct Target Ther). The viral vector approach takes advantage of the natural ability of viruses to infect human cells. Viral pathological genetic sequences are replaced by the desired therapeutic genes and target cells are then infected with the modified viruses, leading to the incorporation of the therapeutic material into the nuclei (Ghoraba et al., 2022 Clin Ophthalmol).

Amongst the viral vectors studied and used for in-vivo gene therapy -which include adenovirus, retrovirus, lentivirus, and herpes simplex virus-, AAV vectors have attracted a significant amount of attention in the field, due to their broad tissue tropism, good safety profile and versatile manufacturing processes. In fact, AAVs are non-pathogenic, do not integrate into the host genome, can sustain long-term gene expression and are often inherently capable of efficient cellular entry thus enhancing transduction efficiency (Wang et al., 2024 Signal Transduct Target Ther).

Notably, the form of AAV used in gene therapy is not the wild-type but a recombinant one (rAAV), which lacks viral DNA and instead contains the recombinant DNA. This will persist as episome in the nucleus of transduced cells and will not be integrated into the host genome, thus it will be diluted over time as cells proliferate and will be eventually lost, which is ideal for some gene therapy applications (Naso et al., 2017 BioDrugs).

rAAVs have the potential to find several applications in the clinic and are currently being tested in clinical trials for a wide range of human diseases, spamming from ocular and neurological to metabolic, hematological, cardiovascular and oncogenic diseases (Wang et al., 2024 Signal Transduct Target Ther). Amongst them, ophthalmology is certainly the main application field of rAAVs. This is due to several reasons: the eye has special immune features that reduce AAV immunogenicity; the eye is small and compartmentalized, thus being easily accessible and requiring low rAAV doses; many ocular diseases are monogenic and thus are suitable for gene therapy.

Gene therapy for ocular diseases was approved by the FDA in 2017 to treat pediatric patients with an inherited retinal disease. Several studies are currently focusing on other possible gene therapies for a wide range of ocular diseases involving from the cornea to the retina (Wang et al., 2024 Signal Transduct Target Ther; Ghoraba et al., 2022 Clin Ophthalmol).

A specialized approach of gene therapy particularly used in the eye is optogenetics. It consists in delivering genetic information that encodes for light sensitive proteins to non-photoreceptor retinal neurons such as ganglion cells, making them sensitive to light stimulation and bypassing the photoreceptors (Ghoraba et al., 2022 Clin Ophthalmol). This strategy could improve vision in patients with Retinitis Pigmentosa (RP) or other inherited diseases where photoreceptors are damaged. The use of optogenetics to restore vision was initially proposed in 2006 by Pan and colleagues (Bi et al., 2006 Neuron). A decade later, the biopharma company GenSight Biologics developed GS030, an optogenetic treatment candidate combining an AAV2-based gene therapy (GS030-DP) with the use of light-stimulating goggles (GS030-MD). As described in the case study below, Axxam was involved in developing and validating the GS030-DP potency assay, as shown in Gael et al., 2018.
Download the poster on GS030-PD

When developing any kind of pharmaceutical, it is mandatory to establish its potency to comply with authorities’ regulations (e.g. FDA or European Medicines Agency, EMA). A potency assay is a quantitative measure of the biological activity of a drug; more specifically, it measures the ability of the product to elicit a specific response in a disease-relevant context. It is used to evaluate product features associated with its quality and manufacturing controls to assure product identity, purity, stability in all phases of clinical studies.

The first step in potency assay development is the choice of the best experimental method, which depends on the mechanism of action of the candidate product. Then, characteristics to be assessed through the assay are the followings: linearity, precision, accuracy, robustness, repeatability, specificity. In fact, potency assays should be able to detect small variations in drug product potency in a robust and specific manner, suitable to assess batch-to-batch variability and drug stability in long-term storage.

Such evaluations can be particularly challenging in case of biological medicines, such as cell and gene therapy medicinal products and tissue engineered products, which have nucleic acid, viral vectors, viable cells and tissues as starting material (Salmikangas et al., 2023 Front Med). For cells, viability and cell phenotype are important features but alone are not sufficient to address biological activity. For example, if cells are transduced with a viral vector, potency is related to the expression of the transgene but also depends on target cells and transduction efficiency in the cells. All these elements should be taken into consideration when developing potency assays.