Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing.

Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the rep gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the rep-cap genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the rep/cap plasmid occurs under several events and rcAAVs spawn from diverse clones.

Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins.

The requirement for robust analytical methods to characterize adeno-associated virus (AAV) vectors is immediate, as the field advances more AAV gene therapies into the clinic and onto commercialization. AAV capsid proteins (VPs) are critical for viral infectivity and vector potency. Thus, complete characterization of the constituent viral capsid proteins of AAV vectors, including their sequences and post-translational modifications (PTMs), is highly recommended to ensure AAV product quality and consistency. Typically, SDS-PAGE analysis followed by in-gel enzymatic digestion and liquid chromatography/tandem mass spectrometry (LC/MS/MS) is used for the characterization of viral capsid proteins. However, due to the limited recovery of digested peptides from the gel, determination of N-terminal sequences of VPs has not been reported to date. In this study, a direct liquid chromatography/mass spectrometry (LC/MS) intact protein analysis was developed to characterize viral capsid proteins in a variety of AAV serotypes. Both N- and C-terminal sequences of six AAV serotypes have been identified based on accurate mass measurement. This method can be used to confirm the identity of AAV serotype and monitor potential capsid protein heterogeneity. Complete sequence confirmation of AAV2 VPs was achieved through LC/MS/MS analysis of peptides generated using multiple enzymatic digestions. LC/MS/MS analysis confirmed the sequences for both N- and C-termini of capsid VPs and revealed acetylation on the N-termini of VP1 and VP3, consistent with LC/MS intact protein analysis.

Generation and characterization of adeno-associated virus producer cell lines for research and preclinical vector production.

Adeno-associated virus (AAV) producer cell lines represent an effective method for large-scale production of AAV vectors. We set out to evaluate and characterize the use of an abbreviated protocol to generate "masterwells" (MWs; a nonclonal cell population) as a platform for research and preclinical vector production. In this system, a single plasmid containing three components, the vector sequence, the AAV rep, and cap genes, and a selectable marker gene is stably transfected into HeLaS3 cells. Producer cell lines generating an AAV2 vector expressing a secreted form of human placental alkaline phosphatase (SEAP) have been created. Several MWs showed vector yields in the 5×10(4) to 2×10(5) DNase-resistant particles/cell range, and the productivity was stable over >60 population doublings. Integrated plasmid copy number in three high-producing MWs ranged from approximately 12 to 50; copies were arranged in a head-to-tail configuration. Upon infection with adenovirus, rep/cap copy number was amplified approximately 100-fold and high yield appeared to be dependent on the extent of amplification. Rep/cap gene expression and vector packaging both reached a peak at 48 hr postinfection. AAV2-SEAP vector was produced in 1-liter shaker culture and purified for assessment of vector quality and potency. The data showed that the majority of the capsids from the MWs contained vector DNA (≥70%) and that purified vector was free of replication-competent AAV. In vitro and in vivo analyses demonstrated that potency of the producer cell-derived vector was comparable to vector generated via the standard transfection method.

Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.

Dual therapeutic utility of proteasome modulating agents for pharmaco-gene therapy of the cystic fibrosis airway.

Pharmacologic- and gene-based therapies have historically been developed as two independent therapeutic platforms for cystic fibrosis (CF) lung disease. Inhibition of the dysregulated epithelial Na channel (ENaC) is one pharmacologic approach to enhance airway clearance in CF. We investigated pharmacologic approaches to enhance CFTR gene delivery with recombinant adeno-associated virus (rAAV) and identified compounds that significantly improved viral transduction while simultaneously inhibiting ENaC activity through an unrelated mechanism. Treatment of human CF airway epithelia with proteasome modulating agents (LLnL and doxorubicin) at the time of rAAV2 or rAAV2/5 infection dramatically enhanced CFTR gene delivery and correction of CFTR-mediated short-circuit currents. Surprisingly, these agents also facilitated long-term (15-day) functional inhibition of ENaC currents independent of CFTR vector administration. Inhibition of ENaC activity was predominantly attributed to a doxorubicin-dependent decrease in gamma-ENaC subunit mRNA expression and an increase in gamma-ENaC promoter methylation. This is the first report to describe the identification of compounds with dual therapeutic action that are able to enhance the efficacy of CFTR gene therapy to the airway while simultaneously ameliorating primary aspects of CF disease pathophysiology. The identification of such compounds mark a new area for drug development, not only for CF, but also for other gene therapy disease targets.