Current Gene Therapy - Volume 5, Issue 3, 2005

This special issue was initially meant to help graduate students entering the field of gene therapy by providing a comprehensive review of recombinant adeno-associated virus (rAAV) from virology to current clinical applications. However, each article is of such a high quality that I believe this issue will also provide a useful document for the entire gene therapy community. Although wild type AAV was studied for decades, Xiao Xiao and Jude R. Samulski's paper in 1996 represents the first evidence that rAAV produced efficient and long-term gene transfer in a mouse after a single in situ administration (i.e. the skeletal muscle) [Xiao et al., 1996]. 1996 was also the year of the lentivirus vector [Naldini et al., 1996]. Yet, 1996 was only one year after the Orkin and Motuslky report [Orkin et al., 1995] emphasizing the need for better vectors! Today progress in rAAV-mediated gene transfer is so spectacular that long-term, efficient, and regulatable transgene expression is reproducibly achieved in large animal models. For example, i) the entire limb of hemophilia dogs can be efficiently transduced resulting in long-term phenotypic correction [Arruda et al., 2004]; ii) rAAV administered once in nonhuman primate muscle shows sustained regulatable transgene expression for more than 6 years [Rivera et al., 2005]. Simultaneously, the discovery of new AAV serotypes along with the ability to encapsidate either “self-complementing” or “single-stranded” vector DNA [McCarty et al., 2001] has turned this vector system into an extremely powerful and versatile tool with preferential organ transduction patterns depending on the AAV capsid origin and/or the vector DNA used. Finally, considerable improvements have been made in the availability of clinical grade rAAV stocks [Snyder et al., 2002], a critical issue, even though large-scale production remains problematic. On behalf of the Current Gene Therapy journal, I would like to thank the authors for their outstanding articles that truly illustrate the impressive number of rAAV-related breakthroughs achieved in the past decade, although with no clear evidence yet that this may be an effective therapeutic approach in humans.

The human parvovirus Adeno-Associated virus (AAV-2) has been classified as a Dependovirus because it requires the presence of a helper virus to achieve a productive replication cycle. Several viruses such as Adenovirus (Ad), Herpes Simplex Virus (HSV), Vaccinia virus, and human papillomaviruses (HPV) can provide the helper activities required for AAV growth. The studies on the helper activities provided by adenovirus have provided useful information not only to understand the AAV-2 biology but also to develop tools for the production of recombinant AAV particles (rAAV). This review will focus on the current knowledge about the helper activities provided by the most extensively studied helper viruses, Ad and HSV-1, and also illustrate the methods used to supply the helper functions rAAV assembly.

The defective parvovirus, adeno-associated virus (AAV), is under close scrutiny as a human gene therapy vector. AAV's non-pathogenic character, reliance on helper virus co-infection for replication and wide tissue tropism, make it an appealing vector system. The virus' simplicity and ability to generate high titer vector preparations have contributed to its wide spread use in the gene therapy community. The single stranded AAV DNA genome is encased in a 20-25 nm diameter, icosahedral protein capsid. Assembly of AAV occurs in two distinct phases. First, the three capsid proteins, VP1- 3, are rapidly synthesized and assembled into an empty virion in the nucleus. In the second, rate-limiting phase, singlestrand genomic DNA is inserted into pre-formed capsids. Our rudimentary knowledge of these two phases comes from radioactive labeling pulse-chase experiments, cellular fractionation and immunocytological analysis of infected cells. Although the overall pattern of virus assembly and encapsidation is known, the biochemical mechanisms involved in these processes are not understood. Elucidation of the processes of capsid assembly and encapsidation may lead to improved vector production. While all of the parvoviruses share the characteristic icosahedral particle, differences in their surface topologies dictate different receptor binding and tissue tropism. Based on the analysis of the molecular structures of the parvoviruses and capsid mutagenesis studies, investigators have manipulated the capsid to change tissue tropism and to target different cell types, thus expanding the targeting potential of AAV vectors.

AAV based vectors can achieve stable gene transfer with minimal vector related toxicities. AAV serotype 2 (AAV2) is the first AAV that was vectored for gene transfer applications. However, the restricted tissue tropism of AAV and its low transduction efficiency have limited its further development as vector. Recent studies using vectors derived from alternative AAV serotypes such as AAV1, 4, 5 and 6 have shown improved potency and broadened tropism of the AAV vector by packaging the same vector genome with different AAV capsids. In an attempt to search for potent AAV vectors with enhanced performance profiles, molecular techniques were employed for the detection and isolation of endogenous AAVs from a variety of human and non-human primate (NHP) tissues. A family of novel primate AAVs consisting of 110 non-redundant species of proviral sequences was discovered and turned to be prevalent in 18-19% of the tissues evaluated. Phylogenetic and functional analyses revealed that primate AAVs are segregated into clades based on phylogenetic relatedness. The members within a clade share functional and serological properties. Initial evaluation in mouse models of vectors based on these novel AAVs for tissue tropism and gene transfer potency led to the identification of some vector with improved gene transfer to different target tissues. Gene therapy treatment of several mouse and canine models with novel AAV vectors achieved long term phenotypic corrections. Vectors based on new primate AAVs could become the next generation of efficient gene transfer vehicles for various gene therapy applications.

In recent years, significant efforts have been made on studying and engineering adeno-associated virus (AAV) capsid, in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest. With our previous knowledge of the viral properties of the naturally occurring serotypes and the elucidation of their capsid structures, we can now generate capsid mutants, or hybrid serotypes, by various methods and strategies. In this review, we summarize the studies performed on AAV retargeting, and categorize the available hybrid serotypes to date, based on the type of modification: 1) transcapsidation, 2) adsorption of bi-specific antibody to capsid surface, 3) mosaic capsid, and 4) chimeric capsid. Not only these hybrid serotypes could achieve high efficiency of gene delivery to a specific targeted cell type, which can be better-tailored for a particular clinical application, but also serve as a tool for studying AAV biology such as receptor binding, trafficking and genome delivery into the nucleus.

Recombinant adeno-associated viral (rAAV) vectors can mediate the safe and long-term correction of genetic diseases in animal models following a single administration. These pre-clinical studies are the basis of human trials that have shown rAAV vector persistence and safety in humans following delivery to lung, sinus, skeletal muscle, brain and liver. Transient disease correction has also been demonstrated in humans treated for hemophilia B and cystic fibrosis using AAV2 vectors. The physiochemical properties of rAAV vector virions are amenable to industry accepted manufacturing methodologies, long-term storage and direct in vivo administration. Recombinant adeno-associated virus vectors are manufactured in compliance with current Good Manufacturing Practices (cGMPs) as outlined in the Code of Federal Regulations (21CFR). To meet these requirements, manufacturing controls and quality systems are established, including 1) adequate facilities and equipment, 2) personnel who have relevant education or experience and are trained for specific assigned duties, 3) raw materials that are qualified for use and 4) a process (including production, purification, formulation, filling, storage and shipping) that is controlled, aseptic, reliable and consistent. Quality systems including Quality Control (QC) and Quality Assurance (QA) are also implemented. These manufacturing procedures and quality systems are designed so the product meets its release specifications to ensure that patients receive a safe, pure, potent and stable investigational drug.

One of the biggest challenges in optimizing viral vectors for gene therapy relates to the immune response of the host. Adeno-associated virus (AAV) vectors are associated with low immunogenicity and toxicity, resulting in vector persistence and long-term transgene expression. The inability of AAV vectors to efficiently transduce or activate antigen presenting cells (APCs) may account for their decreased immunogenicity. AAV mediated gene therapy however, leads to the development of antibodies against the vector capsid. Anti-AAV antibodies have neutralizing effects that decrease the efficiency of in vivo gene therapy and can prevent vector re-administration. Furthermore, recent studies have shown that AAV vectors can elicit both cellular and humoral immune responses against the transgene product. Both cell-mediated response and humoral response to the delivered gene depend on a number of variables; including the nature of the transgene, the promoter used, the route and site of administration, vector dose and host factors. The response of the host to the vector, in terms of antigen-specific immunity, will play a substantial role in clinical outcome. It is therefore important to understand both, why AAV vectors are able to escape immunity and the circumstances and mechanisms that lead to the induction of immune responses. This review will summarize innate and adaptive immune responses to AAV vectors, discuss possible mechanisms and outline strategies, such as capsid modifications, use of alternative serotypes, or immunosuppression, which have been used to circumvent them.

Adeno-associated virus (AAV) vectors exhibit a number of properties that have made this vector system an excellent choice for both CNS gene therapy and basic neurobiological investigations. In vivo, the preponderance of AAV vector transduction occurs in neurons where it is possible to obtain long-term, stable gene expression with very little accompanying toxicity. Promoter selection, however, significantly influences the pattern and longevity of neuronal transduction distinct from the tropism inherent to AAV vectors. AAV vectors have successfully manipulated CNS function using a wide variety of approaches including expression of foreign genes, expression of endogenous genes, expression of antisense RNA and expression of RNAi. With the discovery and characterization of different AAV serotypes, the potential patterns of in vivo vector transduction have been expanded substantially, offering alternatives to the more studied AAV 2 serotype. Furthermore, the development of specific AAV chimeras offers the potential to further refine targeting strategies. These different AAV serotypes also provide a solution to the immune silencing that proves to be a realistic likelihood given broad exposure of the human population to the AAV 2 serotype. These advantageous CNS properties of AAV vectors have fostered a wide range of clinically relevant applications including Parkinson's disease, lysosomal storage diseases, Canavan's disease, epilepsy, Huntington's disease and ALS. Each individual application, however, presents a unique set of challenges that must be solved in order to attain clinically effective gene therapies.

Retinal gene transfer holds big promises for the treatment of inherited and non-inherited blinding diseases, such as retinitis pigmentosa or age-related macular degeneration. Key to the development of successful gene-based therapies for the eye are efficient tools for retinal gene transfer. Vectors based on adeno-associated viruses (AAV) are able to transduce robustly and persistently different retinal cell types of animal models after a single intraocular administration. Recombinant AAV (rAAV) vectors are versatile gene transfer tools in that capsid proteins from dozens of AAV serotypes can be easily interchanged, resulting in the creation of recombinant vectors with unique transduction properties. This has allowed successful proof-of-principle studies using rAAV-mediated gene transfer to restore retinal morphology and function in small and large animal models of retinal diseases. In addition, gene delivery using rAAV vectors in the eye seems to have appropriate biosafety characteristics to rapidly move it from bench to bedside. All the above aspects will be reviewed and discussed in detail below.

Adeno-associated viral (AAV) gene transfer of coagulation factor VIII and IX to skeletal muscle and liver of murine and canine models of hemophilia A and B have resulted in sustained systemic expression and, in several studies, in complete cure of the bleeding disorder. These impressive results prompted initiation of two Phase I/II clinical trials to evaluate the safety of AAV-factor IX gene transfer to muscle and liver of patients with severe hemophilia B. Herein, we have reviewed results from studies in animals with hemophilia, early experience with the vector system in the clinic, recent innovative approaches in vector design and delivery, and strategies to circumvent immunological limitations. Taken together, these studies provide much encouragement for the possibility of future clinical success, but also point out hurdles that still have to be overcome.

Recent studies have shed light on a number of important obstacles to safe and effective gene transfer to the respiratory tract with recombinant AAV vectors. Among these are blocks at the level of receptor binding and internalizations, evasion of proteasomal degradation, inefficiency of nuclear entry, and nuclear factors that inhibit the conversion of rAAV genomes into active double-stranded DNA form. Other important issues have been the size constraints of the vector, the lack of retention of episomal forms of the vector genome, and immune responses which may limit the efficiency of repeated doses of rAAV. Each of these potential obstacles has been addressed with new vector designs. In addition, the availability of an abundance of novel rAAV serotypes, each with its own receptor tropism, has expanded the range of possibilities for long-term success of gene therapy in the respiratory tract.