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Recent clinical successes have propelled recombinant adeno-associated virus vectors (rAAV) to the center stage for human gene therapy applications. However, the exploding demand for high titers of highly pure rAAV vectors for clinical applications and market needs remains hindered by challenges met at the manufacturing stage. The production of rAAV by transfection in suspension cells remains one of the most commonly used production platforms. In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)-mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). Further, we detail a new, robust, and highly efficient downstream purification protocol compatible with both transfection and infection-based harvests that generated rAAV9 stocks of high purity. Our in-depth comparison revealed quantitative, qualitative, and biological differences between PEI-mediated transfection and HSV infection. The HSV production system yielded to higher rAAV vector titers, higher specific yields, and a higher percentage of full capsids than transfection. Furthermore, HSV-produced stocks had a significantly lower concentration of residual host cell proteins and helper DNA impurities, but contained detectable levels of HSV DNA. Importantly, the potency of PEI-produced and HSV-produced rAAV stocks were identical. Analyses of AAV Rep and Cap expression levels and replication showed that HSV-mediated production led to a lower expression of Rep and Cap, but increased levels of AAV genome replication. Our methodology enables high-yield, high purity rAAV production and a biological framework to improve transfection quality and yields by mimicking HSV-induced biological outcomes.

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 1014 (high), 5 × 1013 (medium), or 5 × 1012 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method.

Phenylketonuria is an inborn error of metabolism caused by loss of function of the liver-expressed enzyme phenylalanine hydroxylase and is characterized by elevated systemic phenylalanine levels that are neurotoxic. Current therapies do not address the underlying genetic disease or restore the natural metabolic pathway resulting in the conversion of phenylalanine to tyrosine. A family of hepatotropic clade F adeno-associated viruses (AAVs) was isolated from human CD34+ hematopoietic stem cells (HSCs) and one (AAVHSC15) was utilized to deliver a vector to correct the phenylketonuria phenotype in Pahenu2 mice. The AAVHSC15 vector containing a codon-optimized form of the human phenylalanine hydroxylase cDNA was administered as a single intravenous dose to Pahenu2 mice maintained on a phenylalanine-containing normal chow diet. Optimization of the transgene resulted in a vector that produced a sustained reduction in serum phenylalanine and normalized tyrosine levels for the lifespan of Pahenu2 mice. Brain levels of phenylalanine and the downstream serotonin metabolite 5-hydroxyindoleacetic acid were restored. In addition, the coat color of treated mice darkened following treatment, indicating restoration of the phenylalanine metabolic pathway. Taken together, these data support the potential of an AAVHSC15-based gene therapy as an investigational therapeutic for phenylketonuria patients.

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.