Axxam networking event & special activities
16-20 March 2025 – Milan, Italy
Potency assays for gene therapy CMC studies
Science Spyglass Development of potency assays for clinical studies on gene therapy candidates Gene therapy has emerged as a promising and powerful treatment modality for numerous human diseases, with Adeno-Associated Virus (AAV) vectors recognized as some of the safest and most widely used delivery systems. For these complex biological medicines, where product testing poses unique challenges, potency assays play a critical role in clinical studies and drug licensing. Leveraging Axxam’s advanced technological platforms, these potency assays can be tailored to meet the specific requirements of various application fields, providing robust solutions to this critical step in the drug development process. AAV vectors have been proven to be particularly successful in ophthalmology. In this field, Axxam has recently developed and validated on behalf of GenSight Biologics a potency assay* to support clinical studies of an AAV-based optogenetic drug, i.e. GS030-DP from GenSight Biologics, for visual restoration in patients with Retinitis Pigmentosa. Explore more about gene therapy and potency assays in the expandable boxes below or scroll down to dive straight into our case study on a potency assay for Retinitis Pigmentosa gene therapy. AAVs as a promising strategy for gene therapy 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). AAV-based gene therapy for ocular diseases 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 Potency assays for assessing biological medicines 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
Empowering oncology drug discovery
Science Spyglass Empowering oncology drug discovery with Axxam’s tailored solutions At Axxam, we are committed to advancing oncology drug discovery by applying a range of approaches designed to define molecular targets or disease mechanisms and to identify small molecule hits and lead candidates as starting points for drug development in cancer research. We offer tailored assays and screening solutions, addressing many of the biological mechanisms underlying complex cancer hallmarks: Advanced assays for biomolecular interactions: Our assay designs are based on the current structural understanding of crucial molecular mechanisms driving cancer progression, such as protein-protein or protein-DNA/RNA interactions. This enables us to identify even weakly active small molecules. Biophysical methods for drug interaction validation: We utilize techniques such as thermal shift analysis and microscale thermophoresis to confirm and explore the interactions between compounds and their targets, ensuring precise and reliable results in oncology drug discovery. Covalent binder compound collection: We have recently expanded our chemical libraries to include a library of covalent binders named AXXCovalent, with the aim of identifying lead candidates also for notoriously difficult-to-target proteins. Targeted protein degradation: Axxam has developed assays to explore targeted protein degradation, particularly to address so-called “undruggable” targets like transcription factors and intrinsically disordered proteins. We have also partnered with Symeres to generate a PROTAC (proteolysis-targeting chimeras) platform enabling the identification of degraders that act via the proteasome. Cellular platform techniques: Axxam uses advanced cellular assays to monitor pharmacological effects of compounds on epigenetic and transcriptional mechanisms, splicing processes, and signaling pathways. These assays are performed in physiologically relevant cell types as well as within native cellular environments. High-content screening with 2D and 3D models: Axxam employs high-content screening techniques to monitor changes in the cell phenotypes, both in traditional 2D cultures and more complex 3D tumor spheroids to better simulate the tumor environment. Mass spectrometry-based target identification: Through our collaboration with Momentum Biotechnologies, we employ affinity selection mass spectrometry to identify novel small molecule binders testing our AXXDiversity compound collection, targeting both protein and RNA targets. Contact us Back
Smart cellular assays to study inflammatory skin disorders
Science Spyglass Functional and phenotypic cellular assays to study inflammatory skin disorders Inflammation is a major driver of most chronic skin diseases, causing significant decrease in health-related quality of life for patients. Skin diseases are a heterogenous group of disorders, both acute and chronic, that affect individuals of all ages and are reported to be the most frequent reason for consultation in general practice. The skin is a complex of different cell types providing a physical, chemical and microbiological barrier against external assaults. Keratinocytes represent the major cell type of the epidermis, the outermost layer of skin, and act as the first line of defense of our innate immunity system by sensing pathogens via pattern recognition receptors. Receptor activation, in turn, triggers direct defense mechanisms, like the production of antimicrobial peptides, and release of chemo- and cytokines to recruit and activate additional immune cells. Acute or prolonged dysregulation of keratinocyte function is one of the key steps contributing to the pathogenesis of different types of skin diseases, including atopic dermatitis and psoriasis. Abnormal activation of these cells leads to alterations in their cytoskeleton, expression of cell surface markers (i.e. up-regulation of ICAM-1, reduced expression of E-cadherin and Filaggrin), migration/hyperproliferation of activated cells at the site of inflammation, production and release of pro-inflammatory cytokines and/or chemokines, to maintain a pro-inflammatory state. In fact, impaired resolution of inflammation has been identified as a major culprit in chronic skin diseases. Despite continuous improvement in therapeutic options in recent years, for a many patients affected by chronic skin diseases the response to treatment remains limited. For this reason, there is an urgent need to find novel drugs targeting specific cytokines or receptors implicated in the etiopathogenesis of these disorders. Cell-based models of inflammatory skin diseases With respect to assay development for inflammatory skin disorders, Axxam has now developed and optimized novel cell-based assays to evaluate the activation of human keratinocytes in vitro. These assays are suitable for testing compounds or biologics (antisense oligonucleotides – ASO -, therapeutic antibodies, RNAs) in the early stages of the drug discovery process, as well as to define potential chemical skin-irritants. So far, these validated assays are available in human immortalized (HaCaT) and primary (NHEK) keratinocyte cellular models. These cells are stimulated with a cocktail of pro-inflammatory cytokines, mimicking the inflammatory microenvironment (e.g. TNF-α, IFN-γ) to trigger activation of two major signalling disease-relevant cascades, i.e. the NF-kB (nuclear factor-kappa B) and JAK/STAT (STAT, signal transducer and activator of transcription) pathways. The assays have been miniaturized for 384-well plate formats for compound screening and are designed to analyze 1) cytokine production/release through multiplex measurement; 2) nuclear translocation of inflammation-related transcription factors through immunofluorescence. 1. Cytokine multiplexing assay The cytokine multiplexing assay principle consists of treatment of keratinocytes with a mix of pro-inflammatory cytokines to trigger NF-kB and JAK/STAT pathways, thus leading to production and release of chemokines and cytokines (i.e. IL-6 and CCL5/Rantes), often correlated with the pathogenesis of diseases like atopic dermatitis and psoriasis. The experimental workflow is described in Figure 1. Levels of the two key cytokines CCL5/Rantes and IL-6 released in the medium by activated keratinocyte cellular models are measured by a luminescent readout (AlphaLISA®/AlphaPlexTM, Revvity) which allows simultaneous quantitative determination of two analytes in the same well. Cell culture conditions and pro-inflammatory stimuli are optimized for each cell type to obtain reliable and reproducible CCL5/Rantes and IL-6 levels in physiological conditions. Figure 1: cytokine multiplexing assay workflow. The assay consists of 5 different steps – Step 1: seeding of keratinocytes in 384-well plates; Step 2: stimulation of cells with pro-inflammatory cytokines; Step 3/4: cytokine multiplexing detection in cell culture medium using AlphaLISA®/AlphaPlexTM, Revvity at BMS Labtech Pherastar; Step 5: data analysis. As shown in Figure 2, validation of the assay was performed employing the reference compound Baricitinib, a JAK1/2 inhibitor employed in clinic in the treatment of atopic dermatitis, able to prevent IL-6 and CCL5/Rantes production/release by HaCaT and NHEK cells, stimulated with specific pro-inflammatory cytokine cocktails, in dose-response. Figure 2: Baricitinib treatment inhibits IL-6 and CCL5/Rantes release by HaCaT and NHEK cells stimulated with pro-inflammatory cytokine cocktails. Dose response curves of reference compound Baricitinib for inhibition of production of IL-6 (blu line) and CCL5/Rantes (red line) in cultures of HaCaT (left) and NHEK primary cells (right). Data are presented as Fold Change= Raw values/Central Reference values (referred to stimulated cells not treated with Baricitinib) with a multiplier factor of 100. The developed cytokine multiplexing assay resulted suitable for compound testing in 384-well formats with the aim of identifying novel anti-inflammatory compounds able to inhibit cytokine production/release following keratinocyte activation. 2. Nuclear translocation assay The nuclear translocation assay developed at Axxam allows the detection of the accumulation of NF-kB or Stat1 transcription factors in the nuclei of activated keratinocytes through an imaging-based approach. The experimental workflow is described in Figure 3. Nuclear translocation is measured in HaCaT and NHEK cells stimulated with pro-inflammatory cocktails containing TNF-α and IFN-γ, by immunofluorescent imaging using specific antibodies. Cell culture conditions and stimuli are optimized for each cell type to obtain reliable and reproducible assay signals in physiological conditions. Figure 3: nuclear translocation assay workflow. The assay consists of 5 different steps – Step 1: seeding of keratinocytes in 384-well plates; Step 2: stimulation of cells with pro-inflammatory cytokines; Step 3: immunofluorescence staining with specific antibodies to visualize the targets of interest; Step 4: image acquisition; Step 5: data analysis. As shown in Figure 4, the assay has been validated using the compounds BAY11-7082 (NF-kB inhibitor) and Baricitinib (JAK 1/2 inhibitor), both able to strongly inhibit NF-kB and Stat1 nuclear translocation in HaCaT and NHEK cells, stimulated with a specific pro-inflammatory cytokine cocktail. Figure 4: BAY11-7082 or Baricitinib treatment inhibits respectively NF-kB and Stat1 nuclear translocation in HaCaT and NHEK cells stimulated with a pro-inflammatory cytokine cocktail. A/B) Representative images and dose response curves for BAY11-7082 in HaCaT cells (A) and NHEK cells (B) stimulated with TNF-α and IFN-γ (blue) or treated with vehicle (red, control not stimulated cells); C/D)
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