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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

Science Spyglass High throughput organellar electrophysiology of TMEM175 and TPC2 from freshly isolated lysosomes recorded on the SyncroPatch 384 Application note Axxam S.p.A., MilanNanion Technologies GmbH, Munich Summary Intracellular ion channels are known to play an essential role in various signaling pathways for health and disease, considering that over 80% of transport processes occur inside the cells (1). Among the variety of organellar channels and transporters the proton leak channel transmembrane protein 175 (TMEM175) and the lysosomal two-pore channel (TPC) have received increasing attention in the field given their potential roles in connecting lysosomal homeostasis with pathophysiological conditions such as Parkinson’s disease and cancer (2-4). Consequently, the interest to explore intracellular ion channels as therapeutic targets has grown tremendously indicating a need for high-throughput electrophysiology including patch clamp. There has been some progress in alternative approaches such as solid supported membrane electrophysiology (SSME using the SURFE2R 96SE) recently (5), however, until now, HTS patch clamp has lacked the possibility to collect data from native lysosomes. Axxam and Nanion Technologies have now developed assays to investigate the function and pharmacology of lysosomal channels under native conditions, providing groundbreaking tools for the drug discovery industry. This is possible due to the development of special consumables (single- and multi-hole) dedicated to pursuing organellar recordings in combination with the high flexibility of the SyncroPatch 384 utilizing an ultra-low cell density approach that can use as low as 50k cells/ml, and small volumes of 1 ml for the whole of the 384-well plate, without a drastic reduction in success rate. This can be of extreme importance for expensive – as well as for samples of low quantity (cardiomyocytes, iPS cells or organelles) – to reduce costs and save time. Our approaches resulted in the construction of cumulative concentration response curves and even intraluminal solution exchange during the recording from freshly isolated lysosomes highlighting the broad range of applications possible with the SyncroPatch 384. ResultsTMEM175 Enlarged lysosomes incubated with 1 µM Vacuolin-1 were stained with 0.1 µM LysoTracker™ Red DND-99 (Invitrogen), a red fluorescent dye that stains acidic cellular compartments, such as lysosomes. The dye was added to the cells before isolation of the lysosomes and images were acquired at different magnifications and dilutions using the Operetta system (Perkin Elmer), resulting in an average diameter of 2.1 µm (Figure 1). Figure 1 A – Isolated lysosomes stained with LysoTracker™ Red DND-99 (Invitrogen); images at different magnifications were acquired using the Operetta (Perkin Elmer). B – Average diameter calculated at different dilutions: 3.0 ± 2.1 µm (1:10); 2.0 ± 1.3 µm (1:20); 2.4 ± 1.7 µm (1:50); data are presented as mean ± SD. The remaining lysosomes were used on the SyncroPatch 384 for recording TMEM175 channels expressed endogenously in HEK-293 cells. Critical for success was usage of Nanion’s “Organellar Chips”, a specialized consumable that supported and maintained the integrity of lysosomes throughout the recording and supported cumulative concentration response curves of DCPIB, a novel TMEM175 activator, able to mediate H+ and K+ currents (6) as highlighted in Figure 2. Since TMEM175 channels release luminal H+ into the cytosol, we developed assays using luminal solutions with different pH values, to enhance proton conductance, in addition to potassium flux. The seal resistance in “whole-lysosome” configuration was calculated before compound application and shows average values of 1.4 ± 0.2 GΩ and 2.1 ± 0.6 GΩ at pHluminal 4.0 and 7.0, respectively. TMEM175 activation was accompanied by a drop in Rseal, indicative for stimulation of a leak channel (Figure 2 A). We then executed cumulative concentration additions of DCPIB to activate endogenous TMEM175 channels using only part of the NPC-384 chip (32 wells per condition). Our analysis reveals an EC50 of 65.3 ± 17.5 µM (n=5) at pHluminal 4.0 and 21.5 ± 4.1 µM (n=3) at pHluminal 7.0 for outward currents (ion and proton flux from lumen to cytosol), as shown in Figure 2 D. Representative traces (Figure 2 B-C) clearly show a larger TMEM175 current evoked in the presence of the highest DCPIB concentration in an acidic luminal environment, suggesting enhanced proton flux at acidic luminal pH. Given the known pH dependence of TMEM175 activity (7) we also employed intraluminal solution exchange for the first time where we observed a current modulation after changes in luminal pH. During the experiment with pHluminal 7.0, TMEM175 current was first evoked by DCPIB application, then partially blocked by 4-AP (Figure 3 A). In the presence of 4-AP, acidification of the luminal solution, due to the internal exchange from pHluminal 7.0 to 4.0, increases TMEM175 current (Figure 3 B). A similar experiment was repeated by inverting the luminal pH, starting from 4.0 and changing to 7.0, using the internal perfusion feature of the SyncroPatch 384. In the presence of 4-AP, the reduction of H+ in the luminal solution induces a reduction in TMEM175 current due to a lower proton contribution (Figure 3 C-D). Figure 2 A – Bar graph of seal resistance calculated before and after DCPIB application. B – Representative traces recorded in control and in the presence of increasing concentrations of DCPIB, using luminal solution with pH 4.0, and C pH 7.0. D – Concentration response curve of DCPIB application using different luminal solution, with pH 4.0 (red) and 7.0 (black); in both experiments, cytosolic solutionwas pH 7.0. Figure 3 A – Representative TMEM175 traces recorded in control and in the presence of 100 µM DCPIB (light green) and 2 mM 4-AP (dark green); pHluminal 7.0 – pHcytosolic 7.0. B – Effect of luminal solution exchange (from pH 7.0 to pH 4.0) on TMEM175 current in the presence of 4-AP. C – Representative TMEM175 traces recorded in control and in the presence of 100 µM DCPIB (light green) and 2 mM 4-AP (dark green); pHluminal 4.0 – pHcytosolic 7.0. D – Effect of luminal solution exchange (from pH 4.0 to pH 7.0) on TMEM175 current in the presence of 4-AP. ResultsTPC2 Enlarged lysosomes (Vacuolin, 1 µM) were freshly isolated as described in Schieder et al (8-9) from HEK cells either stably

Science Spyglass Navigating the dualism of the aging and disease landscape with the pertinent tools An interview with Fernanda Ricci, High Content Screening Unit Manager at Axxam. Don’t miss her upcoming webinar: Forever young?Targeting the hallmarks of aging Request link for webinar Read Fernanda’s opinions on the subject of early drug discovery for diseases related to aging: Q. Why do you consider a webinar on aging important? In an era where life expectancy is increasing, the quest to understand aging and promote healthier, longer lives has become more critical than ever. Aging is a complex and multifaceted process that leaves its mark at the molecular, cellular, and systemic levels for all of us, increasing our risk of diseases and disability, and placing demands on health and social care services. However, recent scientific breakthroughs are shedding light on new approaches to unravel the mysteries of aging, such us metabolic regulators, mitochondrial functionality, inflammation pathways. What is truly exciting today is that these discoveries might pave the way for a future where aging is not just a process but a target for intervention; for a future where we may replace the word “aging” with “longevity.” Certainly, we are all committed to reaching a healthier state as much and as fast as possible. The road is still long, with many issues to solve, but starting with the right methods we can speed up the discovery process. For this reason, we are working to establish biological assays relevant to understanding the fundamental processes of aging. Fernanda Ricci, Axxam High Content Screening Unit Manager Q. What tools are you developing to study aging in the laboratory? We are operating on multiple fronts. Aging processes involve several significant molecular pathways, and our commitment extends to miniaturizing all the assays, enabling high-throughput drug screening campaigns to increase the likelihood of finding the right hits for the specific phenotype. Few examples of relevant cell-based assays include DNA damage, mitochondrial dysfunction, autophagy rate readouts, inflammation-based readouts. Q. What are the major relevant pathways or targets involved in aging progression? A real game-changer is certainly inflammation. Chronic inflammation is the troublemaker here, creating the basis for systemic dysfunction that can lead to issues ranging from cancer to heart problems. Some effects at the molecular and cellular levels, for example, involve the mislocalization of transcription factors, consequently altering the genetic script, or a decrease in the stem cell pool despite an increase in senescent cells, and in turn these cells release inflammatory cytokines, adding fuel to the systemic inflammation storm. Remarkably, our vital organelles such as lysosomes and mitochondria get damaged, and they are no longer able to perform their functions, such as clearing cellular toxic products, producing energy, and removing oxidative species. The “Free Radical Theory” of aging comes into play, increasing the probability of protein crosslinking, DNA damage, and a shuffle in gene expression, all contributing to the onset of metabolic and neurodegenerative disorders, as well as cancer in the long term. However, research is progressing rapidly, much like the aging process itself. Several targets, pathways, and phenotypes involved in aging have already been discovered that can be of therapeutic interest and can be addressed selecting the right tools and assays. Today, life science is approaching aging as a Pandora’s box; by treating aging, the incidence of other diseases can also be reduced, achieving a good and longer healthy old age. Aging is inevitable, but how we age could be our decision in the near future. Contact us Back