Joshua C. Grieger

Production and characterization of adeno-associated viral vectors

The adeno-associated virus (AAV) is one of the most promising viral vectors for human gene therapy. As with any potential therapeutic system, a thorough understanding of it at the in vitro and in vivo levels is required. Over the years, numerous methods have been developed to better characterize AAV vectors. These methods have paved the way to a better understanding of the vector and, ultimately, its use in clinical applications. This review provides an up-to-date, detailed description of essential methods such as production, purification and titering and their application to characterize current AAV vectors for preclinical and clinical use.

Phase 1 Gene Therapy for Duchenne Muscular Dystrophy Using a Translational Optimized AAV Vector

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

Packaging Capacity of Adeno-Associated Virus Serotypes: Impact of Larger Genomes on Infectivity and Postentry Steps

ABSTRACT The limited packaging capacity of adeno-associated virus (AAV) precludes the design of vectors for the treatment of diseases associated with larger genes. Autonomous parvoviruses, such as minute virus of mice and B19, while identical in size (25 nm), are known to package larger genomes of 5.1 and 5.6 kb, respectively, compared to AAV genomes of 4.7 kb. One primary difference is the fact that wild-type (wt) AAV utilizes three capsid subunits instead of two to form the virion shell. In this study, we have characterized the packaging capacity of AAV serotypes 1 through 5 with and without the Vp2 subunit. Using reporter transgene cassettes that range in size from 4.4 to 6.0 kb, we determined that serotypes 1 through 5 with and without Vp2 could successfully package, replicate in, and transduce cells. Dot blot analysis established that packaging efficiency was similar for all vector cassettes and that the integrity of encapsidated genomes was intact regardless of size. Although physical characterization determined that virion structures were indistinguishable from wt, transduction experiments determined that all serotype vectors carrying larger genomes (5.3 kb and higher) transduced cells less efficiently (within a log) than AAV encapsidating wt size genomes. This result was not unique to reporter genes and was observed for CFTR vector cassettes ranging in size from 5.1 to 5.9 kb. No apparent advantage in packaging efficiency was observed when Vp2 was present or absent from the virion. Further analysis determined that a postentry step was responsible for the block in infection and specific treatment of cells upon infection with proteasome inhibitors increased transduction of AAV encapsidating larger DNA templates to wt levels, suggesting a preferential degradation of virions encapsidating larger-than-wt genomes. This study illustrates that AAV is capable of packaging and protecting recombinant genomes as large as 6.0 kb but the larger genome-containing virions are preferentially degraded by the proteasome and that this block can be overcome by the addition of proteasome inhibitors.

Single Amino Acid Changes Can Influence Titer, Heparin Binding, and Tissue Tropism in Different Adeno-Associated Virus Serotypes

ABSTRACT Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors.

Adeno-associated virus as a gene therapy vector : Vector development, production and clinical applications

Adeno-associated virus (AAV) has emerged as an attractive vector for gene therapy. AAV vectors have successfully been utilized to promote sustained gene expression in a variety of tissues such as muscle, eye, brain, liver, and lung. As the significance of AAV as a gene therapy vector has been realized over the past years, recent developments in recombinant AAV (rAAV) production and purification have revolutionized the AAV field. It is now possible to produce high yields of vector (10(12)-10(13) genome-containing particles per mL) that are free of contaminating cellular and helper virus proteins. Such vectors have been successfully used in preclinical applications in animal models such as those of hemophilia, lysosomal storage diseases and vision deficiency, all of which have shown therapeutic benefits from rAAV treatment. Clinical trials using rAAV2 for the treatment of hemophilia B, cystic fibrosis, alpha-1-antitrypsin deficiency, and Canavan disease have begun, and reports from these phase I trials support the safety seen in preclinical trials. Eventually, tissue-specific vectors that can potentially evade the immune system will be required to optimize success in gene therapy. In recent years, this has led to the development of retargeted rAAV2 vectors and the identification and characterization of new serotypes from human and nonhuman primates that could potentially achieve these goals. AAV virologists and gene therapists alike have just begun to scratch the surface in terms of the utility of this small virus in a clinical setting. In this chapter, we will provide a comprehensive overview of the recent advances in rAAV vector production and purification, vector development, and clinical applications.

Adeno-associated virus vectorology, manufacturing, and clinical applications.

Adeno-associated virus (AAV) has emerged as an attractive vector for gene therapy. The benefits of using AAV for gene therapy include long-term gene expression, the inability to autonomously replicate without a helper virus, transduction of dividing and nondividing cells, and the lack of pathogenicity from wild-type infections. A number of Phase I and Phase II clinical trials utilizing AAV have been carried out worldwide (Aucoin et al., 2008; Mueller and Flotte, 2008). A number of challenges have been identified based upon data generated from these clinical trials. These challenges include (1) large scale manufacturing technologies in accordance with current Good Manufacturing Practices (cGMP), (2) tissue specific tropism of AAV vectors, (3) high-quality/high potency recombinant AAV vectors (rAAV), and (4) immune response to AAV capsids and transgene. In this chapter, we will provide an overview of AAV biology, AAV vectorology, rAAV manufacturing, and the current status on the latest rAAV clinical trials.

Separate Basic Region Motifs within the Adeno-Associated Virus Capsid Proteins Are Essential for Infectivity and Assembly

ABSTRACT Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 (120QAKKRVL126) and BR2 (140PGKKRPV146) had minor effects on viral infectivity (∼4- and ∼10-fold, respectively), whereas BR3 (166PARKRLN172) and BR4 (307RPKRLN312) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.

Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs

Adeno-associated viral vector 9 (AAV9) has recently been shown to penetrate the blood–brain barrier via intravascular administration, making it a good candidate for diffuse gene delivery. However, the potential side effects of systemic delivery are unknown. Intrathecal viral vector administration may be more invasive than intravenous injections, but it requires far less vector and it can be performed on an outpatient basis, making it an ideal route of delivery for clinical translation. A total of 12 domestic farm pigs (<20 kg) underwent a single-level lumbar laminectomy with intrathecal catheter placement for AAV9 delivery. Animals were perfused and the tissue was harvested 30 days after treatment. Gene expression was assessed by anti-green fluorescent protein immunohistochemistry. Although a single lumbar injection resulted in gene expression limited to the lumbar segment of the spinal cord, three consecutive boluses via a temporary catheter resulted in diffuse transduction of motor neurons (MNs) throughout the cervical, thoracic and lumbar spinal cords. We now present the first successful robust transduction of MNs in the spinal cord of a large animal via intrathecal gene delivery using a self-complementary AAV9. These promising results can be translated to many MN diseases requiring diffuse gene delivery.

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.

Manufacturing of recombinant adeno-associated viral vectors for clinical trials

The ability to elicit robust and long-term transgene expression in vivo together with minimal immunogenicity and little to no toxicity are only a few features that make recombinant adeno-associated virus (rAAV) vectors ideally suited for many gene therapy applications. Successful preclinical studies have encouraged the use of rAAV for therapeutic gene transfer to patients in the clinical setting. Nevertheless, the use of rAAV in clinical trials has underscored the need for production and purification systems capable of generating large amounts of highly pure rAAV particles. To date, generating vector quantities sufficient to meet the expanding clinical demand is still a hurdle when using current production systems. In this chapter, we will provide a description of the current methods to produce clinical grade of rAAV under current good manufacturing practice (cGMP) settings.