Assessing the biopotency of the rAAV9 vector In Vitro

TL;DR

This study developed a reliable in vitro method to assess the potency of AAV9 gene therapy vectors using HuH-7 cells, showing consistent results and long-term stability.

Contribution

A practical and reproducible in vitro assay for AAV9 biopotency using HuH-7 cells is introduced as an alternative to animal models.

Findings

HuH-7 cells reproducibly expressed the transgene, resulting in measurable SGSH production.

Vector genome copies and SGSH expression correlated strongly with viral vector dose (R2 = 0.71–0.95).

rAAV9 vectors remained stable and potent after long-term storage at 2–4°C for over 2.5 years.

Abstract

The potency assay is critical to ensure the effectiveness and consistency of recombinant Adeno-associated Virus (AAV) gene therapy vectors, especially clinical-grade products. AAV serotype 9 (AAV9), known for its neurotropic properties and ability to cross the blood-brain barrier, has been a favored vector for targeting neurogenetic diseases. However, assessing AAV9 biopotency has been challenging due to the insusceptibility of the commonly used cell lines to AAV9. To address this, we utilized a cell-based potency assay using the liver-derived human hepatoma (HuH-7) cell line to evaluate infection by self-complementary (sc)-AAV9 vector expressing human N-sulfoglucosamine sulfohydrolase (hSGSH), currently undergoing evaluation as a potential treatment for Mucopolysaccharidosis (MPS) IIIA. The potency of various scAAV9-hSGSH vector batches was tested in HuH-7 cells which reproducibly expressed the transgene, resulting in measurable SGSH production. The SGSH expression and vector genome copies of various vector batches correlated linearly with the viral vector dose (R2 = 0.71–0.95), indicating a generally strong correlation. The reproducibility of the assay was demonstrated by consistent vector copy numbers and SGSH activity in transduced cells across multiple independent runs. Statistical analysis of the results showed high reliability, with relative intra-assay consistency showing a coefficient of variation (CV) of less than 20%) and inter-assay reproducibility with a CV of less than 25%) affirming the precision of the test. Additionally, our data also demonstrate that long-term (>2.5 years) storage at 2–4°C had no impact on the biopotency of rAAV9 vector confirming long-term stability of the vectors. Hence, we have effectively assessed the biopotency of rAAV9 vector in vitro utilizing HuH-7 cells. Overall, this in vitro assay provides a practical and reliable method to assess AAV9 potency, offering a valuable alternative to animal models and supporting the functional quality and consistency of AAV9 gene therapy vector products in general.

Figures

Quantification of vector genome copy number in HuH-7cell shows effective scAAV9-mediated transduction across various vector batches in a dose-dependent manner.HuH-7 cells were infected with increasing doses from 3750 to 60,000 vg/cell of different batches D, N2, O1 of the LAV139 cGMP vector, Pooled bulk (PB), and Final fill (FF) product. The dose-dependent response to infection was assessed 48 hours post-infection. DNA was extracted from transduced cells and assayed in duplicate by quantitative real-time PCR. Data are expressed as vector genome(vg)/ diploid genomic DNA. NT are non-transduced cells serving as controls. p < 0.01, *p < 0.001 and p < 0.0001 relative to preceding dose.

A dose-dependent increase in SGSH expression in HuH-7 cells transduced with various vector batches confirms effective scAAV9-mediated transduction.HuH-7 cells were transduced with varying doses of different batches of the LAV139 vector- D, N2, O1, Pooled bulk (PB), and Final fill (FF). 48 hours post-transduction, cells were assayed for intracellular SGSH activity. SGSH activity is expressed as units per milligram (U/mg); 1 U = 1 nmol of 4MU released/ 17 hr. NT are non-transduced cells with low baseline SGSH activity. *p < 0.05, p < 0.01, *p < 0.001 and p < 0.0001 vs. corresponding preceding dose.

Dose-dependent Linear Correlation between Vector Doses and the Potency in Transduced Cells.(A) A linear correlation between various vector doses and their corresponding vector genome copy numbers in transduced HuH-7 cells. (B) A positive correlation between different vector doses and the respective SGSH activity in transduced HuH-7 cells. Simple linear regression revealed a dose-dependent effect of the vector on transduction efficiency. The R2 value is calculated for each LAV139 batch (R2 = 0.71-0.95) indicating a positive correlation between the dose and corresponding vector genome copies and SGSH enzymatic activity. Each data point represents the average vector genome copy number (A) and SGSH activity (B) observed at a specific viral vector dose. A solid line joins each data point while a dotted line represents the line of best fit.

Full text

Introduction

Mucopolysaccharidosis type IIIA (MPS IIIA), also known as Sanfilippo syndrome type A, is one of the neuropathic lysosomal storage disorders (LSDs) that result from a deficiency in enzymes responsible for catalyzing Glycosaminoglycans (GAG) [1]. MPS IIIA is an autosomal recessive disorder arising from mutations in the gene encoding N-sulfoglucosamine sulfohydrolase (SGSH). This mutation causes a deficiency in the enzyme heparan N-sulfatase, leading to the accumulation of heparan sulfate, a type of GAG, within lysosomes throughout the body [1,2]. It is a rare and devastating neurodegenerative disorder with an incidence of 1 per 100,000 live births [3]. The prevalence varies among the population in different geographic regions and or ethnic backgrounds, with more prevalence observed in populations where consanguineous marriages are common [4,5]. Consanguineous marriages are common in countries such as Saudi Arabia, Iran, and Gulf states (Middle East), Egypt, Tunisia, Algeria, and Morocco (North Africa), and Pakistan, India, and Bangladesh (South Asia), contributing to a higher incidence of autosomal recessive disorders like MPS IIIA [5]. Individuals affected by MPS IIIA typically develop symptoms within the first few years of life, with the disease primarily affecting the central nervous system, leading to progressive neurodevelopmental regression, severe cognitive decline, behavioral issues, loss of motor skills, and ultimately death in early adulthood [1,6]. Understanding and addressing the unmet needs of MPS IIIA are crucial due to its severe impact on affected individuals and their families.

Gene therapy holds promise for genetic diseases lacking effective treatments or cures by addressing the root cause genetic mutations. Typically, a functional copy of the defective gene is delivered to reestablish the missing enzyme activity. Recombinant adeno-associated virus (rAAV) is favored as a gene delivery vector for its non-pathogenicity, lack of immunogenicity to the host, and broad cell and tissue tropisms [7,8]. The discovery of recombinant AAV serotype 9 (rAAV9)‘s ability to cross the blood-brain barrier (BBB) and target cells within the central nervous system (CNS) further enhances its effectiveness in CNS gene therapy. This capability holds significant promise for the treatment of LSDs and other neurogenetic conditions [9,10]. Notably, we have shown that a single systemic administration of rAAV9 vectors has shown the potential for widespread restoration of enzyme activity, correction of lysosomal storage pathology, and functional neurological improvements in mice with MPS II [11], MPS IIIB [12], and MPS IIIA [13]. To address the existing unmet needs of MPS IIIA, we developed a new self-complementary AAV (scAAV9) gene replacement vector expressing the human SGSH (hSGSH) gene driven by miniature cytomegalovirus (mCMV) promoter [13]. This vector utilizes the validated efficiency of the scAAV [14] and the AAV9’s ability to cross the blood-brain barrier [9,10]. In our preclinical investigations, a single systemic administration of scAAV9-mCMV-hSGSH (via intravenous infusion) in MPS IIIA knockout mice demonstrated long-term expression of SGSH, correction of lysosomal storage in both CNS and somatic tissues, improved behavioral performance, and extended survival [13]. This gene replacement vector is specifically designed for the treatment of MPS IIIA in patients.

For the approval and release of the gene therapy products, the United States Food and Drug Administration (FDA) require the demonstration of a repeatable and reproducible biopotency assay. This assay ensures that each batch of the product possesses the capability to achieve the intended therapeutic effect [15]. The conventional method for evaluating the potency of rAAV vectors involves developing a quantitative biological assay that measures gene transfer and assesses the biological activity of the transferred genetic material within a living biological system. These assays can include in vivo animal studies [16], in vitro assessments using cell cultures [17], or a combination of these methods [18]. While in vivo investigations are essential, responsible practices encourage limiting animal use whenever feasible [15]. In vivo studies are laborious, time-consuming, and suffer from individual variability of animals and the vector administration techniques used by operators. This can be circumvented using cell-based assays monitored to measure the biological function or activity of a therapeutic agent. In vitro potency assays offer advantages over in vivo assays, making them cost and time-effective, efficient evaluations in controlled laboratory settings, and avoiding ethical concerns related to animal testing. Yet, a thorough analysis of the capability of the rAAV9 vectors to quantitatively transduce cells in vitro remains unexplored.

Different AAV serotypes (1–9) are known to exhibit specific tropism with diverse capacities to infect various organs and tissues throughout the body [19]. Systemic administration of AAV9 has been shown to express transgene in a wide range of cell types, including liver, heart, lung, kidney, brain, lung, intestine, skeletal muscle, and testes etc. in both mice [11–13,16] and non-human primates [20]. However, there is limited information on the transduction efficiency of AAV serotypes in cultured cell lines [17]. Currently, no established potency assays are available for AAV9, due to the insusceptibility of fibroblasts, which account for the majority of commonly used cell lines for testing the potency of other AAV serotypes. When tested in vivo in MPS IIIA mice, our test vector, scAAV9-hSGSH exhibited a broad tropism and successfully transduced a variety of cell types, including those in the heart, spleen, lung, intestine, skeletal muscle, kidney, and brain, with particularly high efficiency in hepatocytes [13]. Hence, in this study, we utilized human hepatoma-derived HuH-7 cell lines to measure the biopotency of the rAAV9 vector in vitro. HuH-7 cells have been employed successfully in assessing the potency of the AAV8-hUGT1A1 vector, a gene therapy candidate for treating Crigler-Najjar syndrome [21]. They established methods for quantifying transgenic UGT1A1 expression and biological activity in vitro. Both assays demonstrated a linear dose-response relationship with high reproducibility. Importantly, the in vitro potency results correlated well with in vivo efficacy, suggesting that in vitro assays are reliable for evaluating vector potency and supporting clinical-grade vector release [21].

In this study, we describe a quantitative and validated biological assay that measured the transduction and functionality of the scAAV9-hSGSH gene therapy vector in the transduced HuH-7 cells. The aim was to validate and make accessible the in vitro potency assay for the rAAV9 viral vector, enabling timely and reproducible results in clinical translation, and serving as a model for the characterization of the other rAAV9 gene therapy products.

Materials and methods

rAAV viral vector

The testing article, scAAV9-mCMV-hSGSH viral vector was developed for treating MPS IIIA and previously characterized and tested in a MPS IIIA mouse model [13]. The viral vector genome consists of AAV2 terminal repeats, a truncated 228-bp mCMV promoter, the human SGSH (hSGSH) coding sequence cDNA, and a Simian Virus 40 (SV40) polyadenylation signal. The viral vector was produced at the UNC Vector Core, in compliance with the Current Good Manufacturing Practice (cGMP) enforced by the FDA. The cGMP scAAV9-mCMV-hSGSH vector product (LAV139) was produced in multiple batches (A-Y), using three plasmid co-transfections in a suspension Human embryonic kidney 293 (HEK293) cell line (PCB-293F-01) in 20L Wave bioreactor bags, following previously described procedures [22]. The viral vector was purified via ion exchange chromatography and was dialyzed into 1X PBS (pH 7.0) containing 5% sorbitol and 350 mM NaCl. The titer of the viral vector was determined by dot blotting. Purified products were stored at 4°C before being pooled into the bulk product (PB), upon the completion of production. The PB vector product was transferred to ABL, Inc. for the final sterile fill in Good Manufacturing Practice (GMP) compliance for a planned Phase I clinical trial (IND# 27627). A small portion (1 ml) of Batches D, N2, O1, the pooled bulk product (PB), and the final-filled formulation (FF) were stored at 4°C, as the testing materials for developing the in vitro rAAV9 potency assay in this study. It is important to point out, the vector production took nearly 3 years to complete (10/2020–07/2023), and this study was initiated upon the completion of the production. S1 Table shows the dates of the test article production, pooling, or final sterile-fill.

Cell line

A stable, genetically defined cell model with measurable molecular and functional outcomes was employed, as previously described, to evaluate the biological activity of therapeutics in vitro [23]. In this study, we utilized the human hepatoma-derived HuH-7 cell line to assess the biopotency of the rAAV9 vector. The seed HuH-7 cells were kindly provided by Dr. Charles Askew at UNC-CH. The HuH-7 cells were cultured in DMEM complete growth medium, containing 4.5 g/L D-Glucose, L-glutamine and 110 mg/L sodium pyruvate (Gibco), 10% heat-inactivated FBS (Cytiva), and 1 X Pen/Strep (Gibco).

rAAV viral vector transduction

The HuH-7 cells were expanded in 15 cm culture plates in DMEM complete medium at 37°C (5% CO_2_) and then collected by trypsinization when reaching 80–90% confluency. For viral transduction, HuH-7 cells were seeded at a density of 300,000 cells/well on 6-well tissue culture plates containing 3 mL of DMEM complete medium. The cells were grown for 3 days until the cell confluency reached ~70%. The cells in wells 1–5 on each plate were then incubated with 3 mL DMEM complete medium containing a serially diluted (1:2) rAAV viral vectors (60,000–3,750 vg/cell), while cells in well 6 were incubated with DMEM media only as non-transduced controls. After 48 hours incubation, the cells were harvested by trypsinization followed by washing with 1 mL of PBS. The cells were resuspended in 200 µL of PBS and 100 µL of ddH_2_O before being processed for genomic DNA isolation and SGSH activity assay, respectively. Notably, the assay on each testing article was repeated 2–3 times.

Intracellular SGSH activity assay

To obtain cell lysates for SGSH activity assay, cells suspended in ddH_2_O were processed by repeated freeze/thaw on dry ice and in 37°C water bath 3–4 times, followed by centrifugation at 10,000 rpm for 10 minutes. The supernatants were assayed for SGSH enzyme activity as previously described [24]. The assay quantifies the fluorescent product, 4-methylumbelliferone, generated through the breakdown of the substrate 4-methylumbelliferyl-alpha-D-N-sulphoglucosaminide. The SGSH activity is denoted as unit/mg protein, where one unit corresponds to the release of 1 nmol of 4-methylumbelliferone/17 hours at 37°C.

Quantitative real-time PCR

The cells suspended in PBS were processed to isolate total DNA using QIAGEN QIAamp DNA Mini Kit (Cat # 51306) following the procedures recommended by the manufacturer. The DNA samples were analyzed in duplicate through quantitative real-time PCR, employing PowerUp SYBR Green Master Mix (Thermo Scientific, A25742) and an Applied Biosystems 7000 real-time PCR system, following the manufacturer’s recommended procedures and as outlined in our previous studies [11–13]. TaqMan primers specific to hSGSH were utilized to identify rAAV vector genomes, with forward primer 5’-AAGTCAGCGAGGCCTACGT-3’, and reverse primer 5’-GATGGTCTTCGAGCCAAAGAT-3’. Genomic DNA (gDNA) was quantified simultaneously in parallel samples using human β-actin-specific primers: forward primer 5’-ACCTTCTACAATGAGCTGCG-3’, and reverse primer 5’-CCTGGATAGCAACGTACATGG-3’. Serially diluted vector plasmid and HuH-7 gDNA served as standards. gDNA from non-treated HuH-7 cells were used as controls for background levels and absence of contamination. The data are expressed as vector genome (vg)/ diploid genomic (dg) DNA.

Statistical analysis

Data were analyzed using an unpaired, two-tailed t-test. Data are presented as mean values ± standard deviation. We calculated the coefficient of variation (%CV) for the assay, which is the ratio of the standard deviation (SD) to the mean, expressed as a percentage (SD/mean × 100). Statistical analysis was performed in GraphPad Prism 10 software (GraphPad Software, CA, USA). In all comparisons, statistical significance was established at a threshold of P < 0.05. Significance levels were denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Results

A cell-based in vitro potency assay for rAAV9 gene therapy vector

We hereby evaluated the potency of the rAAV9 vector using a cell-based system. The testing articles were different batches of lot LAV139 which is a cGMP product of scAAV9-based gene replacement vector, scAAV9-mCMV-hSGSH, consisting of a mini-CMV promoter that drives the expression of codon-optimized hSGSH [13]. The cGMP LAV139 vector product was produced in multiple batches (A-Y) of 20 L cell culture Wave bioreactor bags. Purified 26 batches of vector products were stored at 4°C before being pooled into the bulk product (S1 Table). The pooled cGMP vector bulk product was then sterile-filled in Good Manufacturing Practice (GMP) compliance for Phase I Investigational New Drug (IND). To determine the efficiency and reproducibility of the in vitro potency assay, we assessed a selection of the batches D, N2, O1, the pooled bulk product (PB), and the final-filled rAAV9 formulation (FF). The potency of each of these vector batches was assessed in vitro by infecting HuH-7 cell lines with a multiplicity of infection (MOI), defined here as vector genomes (vg) per seeded cell. For each batch, we conducted tests using five different viral doses, ranging from the highest MOI of 60,000 vg/cell to subsequent 2-fold dilutions, with the lowest MOI being 3,750 vg/cell. The cells were harvested 48 hours post-infection for analyses to assess the transgene expression and vector genome (vg) copies in the transduced cells.

Dose-dependent in vitro rAAV9 biopotency in HuH-7 cells

To assess the viral vector transduction efficiency, we measured the vg copy numbers in transduced cells using qPCR for each tested vector product. The results, depicted in Fig 1, illustrate a distribution of the vg in response to the viral vector dose. The vg copy levels consistently exhibited a linear two-fold change through the dilution series of all tested vector batches, indicating a strong positive correlation (R^2^ = 0.85–0.95) between viral doses and vg levels (Fig 3A). Notably, significant vg signals (0.62 ± 0.32) were detected in cells transduced at the lowest viral vector dose, while no meaningful vg signals (0.005 ± 0.004) in non-transduced control cells (p < 0.0001) (Fig 1). These data effectively showed that scAAV9 can transduce HuH-7 cells in vitro, in a dose-dependent manner, and a quantifiable vector genome signal was present in the transduced cells, even at the lowest MOI for each batch.

*Quantification of vector genome copy number in HuH-7cell shows effective scAAV9-mediated transduction across various vector batches in a dose-dependent manner.HuH-7 cells were infected with increasing doses from 3750 to 60,000 vg/cell of different batches D, N2, O1 of the LAV139 cGMP vector, Pooled bulk (PB), and Final fill (FF) product. The dose-dependent response to infection was assessed 48 hours post-infection. DNA was extracted from transduced cells and assayed in duplicate by quantitative real-time PCR. Data are expressed as vector genome(vg)/ diploid genomic DNA. NT are non-transduced cells serving as controls. **p < 0.01, ***p < 0.001 and ***p < 0.0001 relative to preceding dose.

Next, to further assess the potency of the tested vector product, we assayed the cell lysates for SGSH activity to determine the expression and biological activity of the transgene product. The SGSH expression was detected in all cells transduced with the tested AAV9 vector products. Non-transduced (parental) cells showed minimal endogenous expression of SGSH (6.2 ± 1.74), while at the lowest vector dose (3,750 vg/cell), all transduced cells exhibited higher SGSH activity than non-transduced cells. Specifically, significantly higher SGSH activity was observed in cells transduced with three tested vector batches- D, O1, and FF (Fig 2). The higher activity levels in transduced cells, even at the lowest dose, suggest that the vectors are effective in achieving significant expression of SGSH. Further, the SGSH activity linearly associated with viral dilutions, showing a positive correlation between viral dosage and transgene expression (R^2^ = 0.71–0.88) (Fig 3B). This indicates that the transduction with the tested AAV9 vector products successfully increased SGSH expression and biological activity in the cells, demonstrating the potency of the vector.

*A dose-dependent increase in SGSH expression in HuH-7 cells transduced with various vector batches confirms effective scAAV9-mediated transduction.HuH-7 cells were transduced with varying doses of different batches of the LAV139 vector- D, N2, O1, Pooled bulk (PB), and Final fill (FF). 48 hours post-transduction, cells were assayed for intracellular SGSH activity. SGSH activity is expressed as units per milligram (U/mg); 1 U = 1 nmol of 4MU released/ 17 hr. NT are non-transduced cells with low baseline SGSH activity. *p < 0.05, **p < 0.01, ***p < 0.001 and ***p < 0.0001 vs. corresponding preceding dose.

Dose-dependent Linear Correlation between Vector Doses and the Potency in Transduced Cells.(A) A linear correlation between various vector doses and their corresponding vector genome copy numbers in transduced HuH-7 cells. (B) A positive correlation between different vector doses and the respective SGSH activity in transduced HuH-7 cells. Simple linear regression revealed a dose-dependent effect of the vector on transduction efficiency. The R2 value is calculated for each LAV139 batch (R2 = 0.71-0.95) indicating a positive correlation between the dose and corresponding vector genome copies and SGSH enzymatic activity. Each data point represents the average vector genome copy number (A) and SGSH activity (B) observed at a specific viral vector dose. A solid line joins each data point while a dotted line represents the line of best fit.

Reproducibility of the in vitro AAV9 potency assay using HuH-7 cells

To assess whether the assay we utilized was reproducible and consistent, the precision of the test was assessed via intra-assay and inter-day assay variability. The coefficient of variation (CV) or Relative Standard Deviation (RSD) quantifies assay repeatability by expressing relative variability as a percentage: [Standard deviation/ Mean) * 100%]. We calculated the %CV of our assays based on the expressed vg copy levels and SGSH activity in the transduced cells, respectively. For the intra-assay variability, we conducted two assay replicates for each dose of the LAV139 vector batch (ranging from 3,750–60,000 vg/cell). These replicates represent independent experiments performed on the same day. The mean and standard deviation (SD) for each dose were calculated from these replicates to determine the %CV for each assay. The mean intra-assay %CV was then calculated, as shown in Tables 1 and 2. The inter-day assay precision was analyzed by performing the assay on 2 or 3 independent days to evaluate the variability of the potency assay across different days.

Table 1: Intra-assay variability of HuH-7 cells transduction measured by qPCR.

Table 2: Intra-assay variability of HuH-7 cells transduction measured by SGSH activity.

The mean intra-assay %CV for the vector batches tested based on vg copy numbers quantified via qPCR at the lowest dose ranged between 5.82%−40.85% (Table 1). Except for batch N2, the %CV for other batches at a dose of 3,750 vg/cell was ≤ 20%. The %CV varied among batches and increased at higher dose groups (Table 1). The intra-assay %CV based on the SGSH activity was ≤ 15% at the lowest dose and showed a satisfactory %CV of ≤22% for the higher doses across vector batches tested (Table 2). The Intra-assay %CV was higher for calculations based on qPCR compared to the SGSH activity indicating that the variability varies greatly depending on the specific assay being performed.

We then assessed the inter-day assay precision by calculating the %CV for each vector dose across 2 independent experiments performed on different days. The inter-day %CV based on qPCR exhibited variability across different doses and batches ranging from 5%−53% (Table 3). While the inter-day assay precision based on SGSH activity ranged from 1.2% −45% (Table 4). For every batch examined, the inter-assay %CV based on SGSH activity was at its minimum with the highest vector dose compared to all other doses (Table 4). Examining the final formulated LAV139 vector and conducting the assay on different days showed mean intra-assay and inter-assay CV within the acceptable threshold of <25%. These findings validate the repeatability of the assay in reliably detecting vg copy levels and SGSH enzyme activity in the transduced cells.

Table 3: Inter-day potency assay precision measured by qPCR.

Table 4: Inter-day potency assay precision measured by SGSH activity.

Stable potency of rAAV9 vector stored at 4°C

To assess the stability of rAAV9 potency using HuH-7 cells, we compared 3 batches of the scAAV9-hSGSH (D, N2, O1), pooled bulk product (PB) and final-filled product (FF), which were produced over a period of nearly 3 years and stored at 4°C (S1 Table). In general, the results showed no significant differences in SGSH activity levels in cells transduced with the tested vector batches, especially at 3 lower vector doses (S1A Fig), which were significantly higher than the endogenous SGSH activity in non-transduced cells (Fig 2). Further, the vg levels varied slightly among cells transduced with different batches D, O1, PB and FF vector product, especially at the 3 low doses (S1B Fig). Notably, cells transduced with batch N2 showed higher vg levels than cells transduced with other tested vector batches (S1B Fig), probably due to greater assay variations. Importantly, we did not detect significant differences in SGSH activity and vg levels among cells transduced with batches D, O1, PB and FF products (S1 Fig). Comparing the initial vector batch stored at 4°C (batch D) with the final formulation (FF) revealed no significant differences in SGSH activity at the four lower doses (S1A Fig) or in vg copy numbers (S1B Fig), indicating that our vector remained stable under refrigerated conditions. These data indicate that long-term storage at 4°C have no impact on the biopotency of the tested scAAV9-hSGSH vector product.

Discussion

Due to the limited infectivity of AAV9 to fibroblast cells cultures in vitro [17], the use of cell-based potency assays as a measure of therapeutic efficiency poses a significant challenge for the translation of AAV9 gene therapy vectors for treating human diseases. In this study, we utilized a HuH-7 cell-based in vitro potency assay enabling rapid and effective potency testing of an AAV9 gene therapy vector. This potency assay evaluates transduction efficiency and quantifies the biological activity of the target transgene product, rSGSH, of the cGMP scAAV9-mCMV-hSGSH vector product.

For the AAV vector potency determination, the effectiveness depends on multiple steps such as infection (delivery), genome conversion/transcription, and transgene expression/function. The rAAV vector undergoes a multistep path to reach the nucleus of the host cell, the release of the genome from the capsid, and the expression of the transgene involving numerous host factors [25,26]. Notably, the presence of the vector genome in the transduced cells does not necessarily ensure expression of the transgene product [26]. Therefore, to assure potency at each step, we devised an in vitro assay that independently assesses infection by quantifying the vector genome copies in the transduced cells and the subsequent rSGSH expression measured through enzymatic activity of the target SGSH protein. We tested different batches of LAV139 vector product by administering 5 doses of the viral vector, beginning with an MOI of 60,000 viral genomes per cell, and then proceeding with 2-fold dilutions in cell-based assays. While an MOI of 60,000 vg/cell may seem like a high starting point, MOIs ranging from 10,000 to as much as 500,000 vg/cell have been used previously for human gene targeting [27,28]. AAV9 serotype at an MOI of 100,000 viral genomes/cell was not efficient in transducing several human cell lines [17]. We demonstrated that in scAAV9-mCMV-hSGSH-transduced HuH-7 cells, both the vector genome copies (Fig 1) and the SGSH activity (Fig 2) were detectable even at a low AAV9 dose (3750 vg/cell) for all batches tested. The vector genome copy numbers exhibited a linear two-fold dose-response relationship across the viral dilution series (Fig 3A). This was expected due to the sensitivity of quantitative PCR, which targeted the transgene sequences of the vector genome, with calibration performed using dilutions of genomic DNA and a housekeeping gene (standard curve). This allowed for the absolute quantification of the average count of vector genome copies in the transduced cells. In contrast, the dose-response relationship for SGSH activity was non-linear, though reproducible, over the dilution range, possibly due to the complexity of the enzyme-substrate interactions and the fluorescent product of the assay [29].

The precision of the in vitro potency was assessed by calculating the coefficient of variation (CV) based on the variability of replicates. Although an international standard is lacking, for cell-based potency assays, the acceptable %CV is set at 10–30% [30]. We assessed intra-assay and inter-day assay variability, and the mean %CV for the transduction efficiency of the final vector formulation met the acceptable criteria (below 25%). The intra-assay %CV showed higher variability with qPCR compared to SGSH activity, suggesting that the variability may be influenced by the specific type of assay and the measurement method. Inter- and intra-assay variation (repeatability) depends on various factors and ensures consistency by allowing the same measurements or experiments to be performed multiple times under the same conditions. This consistency reduces the variability between measurements, leading to a lower CV. Overall, batch LAV139 D and the final filled rAAV formulation when tested over three independent days, showed lower CVs based on the vg copy levels and intracellular SGSH activity assay, suggesting that repeatability of tests is essential for satisfactory precision. It is necessary to increase the number of replicates of the assays depending on the availability of vector material and the costs involved. Moreover, to ensure consistent assay performance and comparability, we need reference standards, controls, and acceptance criteria which must be established, maintained, and characterized [15]. Our described potency assay currently utilizes a 6-well plate format. While effective, this may restrict the number of samples that can be processed simultaneously. Adopting higher-density plates, such as those with 24–96 wells, could significantly enhance the assay’s throughput and replication capacity, allowing for more efficient and extensive testing.

AAV vector production was conducted in multiple batches, which were combined and dispensed into clinical vials. Ensuring consistency among vector batches and lots for clinical trials is crucial, as it ensures reliability, accuracy, and reproducibility of the trial outcomes. Consistency guarantees that the gene therapy vector maintains uniformity in its properties, potency, and efficacy across different batches or lots produced [15]. Overall, vg copy levels slightly varied among batches, but the SGSH activity at the lower doses had no significant differences (S1 Fig). Importantly, the mean vg copy levels and SGSH activity in the cells receiving the different viral vector doses were comparable between the pooled bulk and the final filled batches, suggesting no loss of biological activity through storage and processing (S1 Fig). It is crucial to maintain vector stability and functionality throughout the manufacturing process and storage to attain optimal function. Previous studies have assessed the effects of factors such as temperature, freeze-thaw cycles, and storage conditions on the integrity and transduction efficiency of AAVs [31,32]. Under refrigerated storage conditions, the AAV-based vector was shown to lose efficacy [32]. However, our data in this study showed that the long-term (≥2 years) storage of the scAAV9-hSGSH viral vectors at 4°C did not affect the transduction efficiency when tested in vitro, demonstrating the long-term stability of rAAV9 vector at 4°C. The diminished AAV vector stability under 4°C observed in previous studies [32] might be due to differences in factors such as manufacturing processes and vector formulation. The long-term stability of rAAV9 at 4°C demonstrated in this study may address a critical issue in clinical application and commercialization of rAAV9 gene therapy products, regarding retaining the AAV biopotency during the vector transportation and storage

In recent years, the numbers of AAV vector-based clinical trials have increased steadily. Numerous AAV-based gene therapy products are undergoing testing from the laboratory to clinical settings [33]. AAV vectors have been the preferred choice for gene therapy research, leading to the FDA-approved treatments, including Luxturna for Leber Congenital Amaurosis 2 (LCA); Hemgenix and Beqvez for Hemophillia B, Upstaza for aromatic L-amino acid decarboxylase deficiency, Elevidys for Duchenne Muscular Dystrophy; Roctavian for Hemophilia A [34]. The demonstrated trans-blood-brain-barrier-neurotropic AAV9 is a commonly utilized serotype in gene therapies especially for neurological diseases, with the FDA- approved Zolgensma for Spinal Muscular Atrophy (SMA), a severe neurogenetic disorder [35]. Establishing reliable and standardized potency measurements will expedite the release of therapeutic products, enabling a smooth transition into clinical trials and market approval. In conclusion, we have successfully employed a reliable, cost- and time-efficient assay to assess the in vitro potency of an AAV9-based gene therapy vector product using HuH-7 cells. This HuH-7 cell-based assay may be broadly applicable to biopotency assessment of all rAAV9 gene therapy vector products.

Supporting information

S1 TableStatus of scAAV9-hSGSH vector products (LAV139)*.This table summarizes the production dates and storage conditions of individual vector batches, including intermediate pooled bulk (PB) and final filled (FF) products. All batches were stored at 4°C and assayed for potency after pooling.(DOCX)

S1 FigComparative assessment of potency of LAV139 batches at a given vector dose.The HuH-7 cells were transduced with increasing doses of the scAAV9-hSGSH vector batches, LAV139 D, N2, O1, PB, FF. The cells were analyzed for rSGSH expression by SGSH activity assay and vector genome (vg) by qPCR. (A) Comparison of SGSH activity levels in cells transduced with vector batches at a given dose. (B) Comparison of vector genome levels in transduced cells at the given vector dose. ns: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001.(PDF)