Introduction

Gene therapy (GT) is an innovative approach designed to modify gene expression in targeted cells or modulate a biological function through various strategies [1]. Numerous GT-based drugs have been developed to treat a wide range of diseases that are related to disorders in gene expression in different types of cells [1]. One of the cutting-edge GT techniques is the Adeno-associated virus (AAV) that has shown promising results in treating complex diseases, including cancer, cardiovascular diseases, respiratory disorders, and CNS disorders [2]. Despite all the recent improvements in AAV development, there are still limitations and challenges in AAV vector manufacturing and analytical processes [3].

One of the main issues is inadequate lab space [4]. Many academic institutes and companies cannot dedicate enough space for manufacturing cleanrooms, cell culture rooms, and other necessary equipment for AAV vector development [4,5]. Moreover, AAV production on a large scale requires extensive purification systems and bioreactors [5]. As a result, many laboratories lack sufficient capacity to accommodate such equipment [4,5]. Another issue is that AAV production has a complex workflow with multiple steps, including vector generation, purification, and quality control [6]. Insufficient lab space cannot support complex workflows, leading to inefficient AAV production [4].

Another problem is a shortage of expertise [7]. AAV manufacturing is a multi-step process that demands highly qualified personnel with comprehensive knowledge in gene therapy to produce efficient AAVs for commercial and clinical use [7]. Lack of expertise may lead to delays in AAV production, purification, and quality control [7].

Technical challenges are also considerable bottlenecks to viral vector production [7]. One issue is delays in cell-based production, which includes designing custom cell lines for viral production, assay development for cell-based testing, cell-based purification, and scale-up of cell culture systems [8]. Another issue is limitations in testing for full and empty capsid, which includes steps such as sample preparation, quantification analysis, imaging to test capsid integrity, and quality control [9]. These technical issues make AAV production a complex, time-consuming, and expensive process [10].

This article provides an overview of AAV vectors and explains testing and manufacturing processes. Also, current challenges in AAV production will be outlined focusing on how Emery Pharma, as a contract research organization (CRO), is able to provide solutions to these technical and clinical issues and therefore contribute to enhancing AAV vector production.

Adeno-associated viral vectors in the lab: structural features and related testing methods

AAV was first identified as contamination during simian Adenovirus (Ad) preparation in 1965 [15,16]. These viruses are named adeno-associated since their function is dependent on the presence of a helper virus (I.e., herpesvirus) to infect targeted cells [16,17]. AAV belongs to the Dependovirus genus within the Parvoviridae family which can be found in various vertebrates such as non-human primates (NHPs) and humans [2]. These viral vectors contain a ~4.7kb single-stranded DNA genome [18]. The genome is flanked by T-shaped inverted terminal repeats (ITRs) on both ends which have an essential function in the viral life cycle (i.e., replication and packaging) (Figure 1) [14]. In addition, AAV vectors contain three viral proteins VP, VP2 and VP3 with a 1:1:10 ratio in order [14].

Figure 1. The genomic structure of AAV. a) The capsid surface showed blue to green to red, then yellow from the center to the surface, respectively. The white triangles display asymmetric units within the virus bounded by a 5-fold axis (5f) and two 3-for axes (3f) segregated by a 2-fold axis (2f) line. DE loop, HI loop, VR-VII, and VR-IV may vary within AAV serotypes. b) cross-section of AAV, dark blue regions show β-strand and β-sheet as secondary structure elements. c) ssDNA with about 4.5 kb capacity flanked by ITRs. P5 and p19 are promoters expressing Rep78, Rep68 and Rep52, Rep40 (regulatory proteins) respectively while p40 regulates VP1, VP2 and, VP3. AAP is an assembly-activating protein [14].

All these structural features of AAV play a crucial role in capsid development [2]. As mentioned, AAV depends on a helper virus to effectively infect the targeted cells [2]. For this reason, specific cell lines such as HEK293 cells and Sf9 cells or plasmid-based systems (e.g., Rep-Cap plasmid, transgene plasmid and, helper plasmid) are used to simulate helper virus function which is essential for AAV replication and packaging [15]. Co-infection by a helper virus should be performed carefully to avoid any contamination while producing AAV vectors in high yields [16].

One important factor for an AAV product to meet safety requirements is to be tested for helper virus contamination [16]. Since one of the primary risks of contamination by helper viruses is infection and immune response, AAV products should be tested through cell-based assays such as antigen-capture ELISA or PCR techniques to detect residual proteins or helper virus DNA [17].

ITRs are another essential component of AAV, which ensures the packaging and stability of the AAV genome within the capsid [18]. ITRs form a palindromic hairpin structure that functions as a signal for genome recognition [18,19]. Incomplete or damaged ITRs disrupt the palindromic structure [18]. As a result, genome recognition signaling will be interrupted and lead to inefficient packaging [18]. Another role of ITRs is to protect the viral genome from nuclease degradation and facilitate its proper integration into the targeted cell [18]. Damaged ITRs cannot protect the viral genome from degradation, leading to its instability in the host cell [18]. Therefore, to ensure the integrity and functionality of ITRs, techniques such as Quantitative PCR (qPCR) or sequencing should be used [18].

Viral proteins (VPs) play a vital role in infectivity and stability of the vector since they form the capsid’s outer shell and assist in cell attachment [14]. Any damage to VP proteins results in disrupted cell binding and capsid vulnerability to physical stress and enzymatic degradation [20]. Hence, analytical methods such as capillary electrophoresis, SDS-PAGE and Liquid Chromatography Mass Spectrometry (LC-MS) should be performed to monitor and confirm the existence of VPs and their ratio within the capsid [21,22].

Another vital component of the AAV capsid is a Single-Stranded DNA Genome (ssDNA) that limits the therapeutic gene payload to ~4.7kb [2,14 ]. This means any regulatory sequence or necessary elements, such as enhancers and promoters, must fit within this small size [2,14,23]. Therefore, this limitation necessitates an accurate design and assembly of the targeted gene to ensure that all therapeutic elements are accommodated within the AAV capsid [23].

Damage or mutation in ssDNA may result in insufficient gene delivery to the targeted cell [24,25]. Also, fragmented ssDNA is more exposed to nuclear degradation and less stable within the host cell [24,25]. To test the efficiency of the inserted gene, methods such as Digital Droplet PCR (ddPCR) or qPCR can be used [26]. AAV components, their importance, the effects of damage to each subunit as well as testing methods are summarized in Table 1.

Table 1. Summary of AAV components and evaluation methods.

Emery Pharma is a CRO that offers a wide range of laboratory techniques to access such integrity and efficiency assays for AAV capsid components [27]. One of these techniques is LC-MS, which is a useful method to test the presence of VP proteins and their standard ratio in a capsid [21,27]. Also, full and empty capsid analysis, which is important for therapeutic efficacy can be assessed using mass spectrometry methods [28]. Furthermore, the high sensitivity of LC-MS makes this technique a useful approach for impurity detection in AAV product [29]. Therefore, Emery pharma is able to assist pharmaceutical companies to evaluate AAV products and their therapeutic potency through innovative techniques and professional expertise [27].

Post-Translational Modifications (PTMs)

PTMs are covalent processes that modify a protein’s characteristics through proteolytic cleavage or binding chemical groups such as methyl, acetyl, glycosyl, and phosphoryl [21,30]. Due to these chemical modifications, PTMs can significantly affect AAV function within the host cell via changing protein structure and dynamics [21,30]. Thus, PTMs should be carefully monitored for in-depth analysis of AAV VP proteins to characterize different VP proteoforms and their potential effects on AAV product safety and durability [21]. Table 2 provides a list of PTMs and their effects on AAVs function.

Table 2. Types of PTMs on AAV capsids and their effect on AAVs functionality. (+) indicates the addition of one chemical group to another [30]. (NLS) stands for Nuclear Localization Signals and SUMO is the abbreviation of small ubiquitin-like modifiers.

As shown in Table 2, PTMs can enhance or suppress the delivery of therapeutic genes by AAVs. Moreover, chemical modifications such as acetylation and glycosylation may help AAVs to escape host immune response. On the other hand, some types of PTMs may negatively affect AAV capsids' stability and delivery efficiency. For instance, ubiquitination in capsid proteins can mark them for proteasomal degradation and lower their functionality.

Testing methods for PTMs and their influence on AAVs can be performed using mass spectrometry, western blotting, and chromatography [21,31]. Emery Pharma offers LC-MS and LC-UV methods, which can verify the primary amino acid sequence and identify PTMs (e.g., glycosylation) in these peptides [27]. Moreover, recent studies have illustrated the efficacy of LC-MS and techniques in full-length sequencing of AAV VP proteins and detecting PTMs on them [21,22].

For example, Smith et al.,2023 designed a study to evaluate PTMs on AAV2 as Critical Quality Attributes (CQAs) to assure the quality of AAV products (Figure 2) [21]. To identify VPs within AAV vector, they used Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry (HILC-MS) along with difluoroacetic acid, a mobile phase modifier, to achieve a clear separation of all 3 AAV VPs [21]. This method showed high-quality mass spectrometry data for AAV identification [21]. Scientists were able to characterize 18 PTMs during AAV2 peptide mapping, including acetylation, deamidation, and phosphorylation [21].

Figure 2. An illustration of the PTM testing workflow generated for PTM monitoring and rapid AAV serotype characterization [21].

Challenges related to AAV vectors

Despite all the benefits, there are manufacturing and lab space limitations as well as a shortage of expertise and clinical challenges that need to be addressed to optimize AAV production, function, and effectiveness.

Lab space limitations and shortage of expertise

As mentioned, constraints in lab space and expertise remain a challenge in the AAV production workflow. To address limitations in lab space, institutions can delegate to contract research organizations to share cleanrooms and bioproduction facilities [27,31]. Moreover, scalable and modular production platforms such as single-use bioreactors and closed system workflows are needed to enhance spatial utilization on clinical and commercial scales [32].

To mitigate the shortage in expertise, it is important to expand educational initiatives in gene therapy manufacturing and encourage cross-disciplinary training to build a skilled workforce [33]. Moreover, outsourcing key analytical and manufacturing processes to Emery Pharma, a CRO with a team of qualified scientists in bioanalytical and molecular biology, is a key approach to overcome the shortage in workforce [27,33].

Manufacturing challenges

The viral vector manufacturing process includes upstream, downstream, viral vector formulation, and fill/finish (Figure 3) [14]. Upstream involves plasmid development and production, cell expansion to expand the cell density, plasmid transfection to introduce the plasmid to the cells following cell expansion, and vector production in plasmid-transfected cells [14]. Downstream is a purification workflow and formulation, fill/finish aimed to preserve the stability and functionality of the vector during manufacturing and storage [14].

In the upstream workflow, purity of plasmid DNA and the use of adherent cells for cell expansion may have contamination risks [14]. In the downstream process, filtration, which is the most expensive step, purification, and cell lysis are the main concerns [14]. Lastly, minimizing degradation in AAV vectors and finding a suitable condition to store gene therapy products are some of the challenges of formulation, fill/finish [14].

Degradation mechanisms in AAVs can be divided into physical and chemical, which can affect the quality of AAV products during storage, shipping, and handling [14]. Surface adsorption, aggregation, and unfolding of the AAV structure during expression or production are known as physical degradation, while chemical degradation refers to incorrect disulfide bond formation, deamidation, and oxidation of capsid proteins, as well as iso-aspartic acid formation in proteins known as Isomerization.[14].

To manage degradation and ensure product quality during AAV production, in-process testing is necessary. Emery Pharma offers a wide range of analytical techniques to manage and mitigate both chemical and physical degradation in AAV products [14,27]. For example, to monitor and prevent chemical degradation, this company provides LC-MS testing to identify capsid PTMs as well as HPLC and peptide mapping for detailed analysis of capsid integrity [27]. Moreover, Emery Pharma assists in physical degradation monitoring through Size-Exclusion Chromatography (SEC) and Dynamic-Light Scattering (DLS) to detect unfolding or aggregation in AAV capsids [27].

Figure 3. An overall view of the AAV vector manufacturing process [14].

FDA requirements

AAV-based drugs must undergo a comprehensive evaluation to gain FDA approval [34]. The first step is to conduct pre-clinical research to prove the safety and effectiveness of the product [34]. The next step is to submit an investigational new drug application (IND), which involves pre-clinical research data, to the FDA before initiating clinical trials [34]. Following the IND application, clinical trials start in 3 phases: phase 1, which includes safety assessment and is performed on a small number of patients [34]. Phase 2 is preliminary safety and efficacy, and lastly, phase 3 covers confirmatory safety and efficacy [34]. After passing clinical trials, a Biologics License Application (BLA), which includes both pre-clinical and clinical data, should be submitted to the FDA [34]. Thereupon, the provided data is fully reviewed to approve the safety and efficacy of the AAV vector, then, based on the results, the FDA issues a Complete Response Letter (CRL), which allows the gene therapy product to be marketed [34]. After the final approval, post-approval monitoring will continue to ensure the safety and efficacy of the AAV-based gene therapy product [34].

Conclusion

Adeno-associated viral vectors are a revolutionary therapeutic approach offering multiple serotypes and a vast tropism. Despite all the advantages, the complexity of AAV production, analytical assessments, and regulatory compliance remains the main impediment in the vector production workflow, which underscores the role of contract research organizations. Emery Pharma, as a CRO, stands out as a key partner in the development of AAV-based therapies with its specialized expertise and advanced laboratory infrastructure. Therefore, from method development and bioanalytical analysis to clinical trials and regulatory support, Emery Pharma facilitates the transition of AAV products from bench to bedside and plays a vital role in advancing both scientific research and industry progress within the gene therapy pipeline.

About the Author

Originally authored by Viana Keyvan, an Emery Pharma Intern.