Chimeric herpes simplex virus/adeno-associated virus amplicon vectors

Chimeric or hybrid herpes simplex virus type 1/adeno-associated virus amplicon vectors combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and stable transgene expression of AAV. These chimeric vectors have been demonstrated to support transgene expression for significantly longer periods than standard HSV-1 amplicons. Moreover, HSV/AAV hybrid vectors can mediate integration at the AAVS1 pre-integration site on human chromosome 19 at a relatively high rate, although random integration has also been observed. One major remaining hurdle of HSV/AAV hybrid vectors is the low packaging efficiency and titers when AAV rep sequences are included in the amplicon vector. In the conditions prevalent during the replication/packaging of HSV/AAV hybrid amplicons into HSV-1 virions, in particular the presence of HSV-1 replication factors and AAV Rep protein, at least three different viral origins of DNA replication are active: the HSV-1 ori, the AAV inverted terminal repeats (ITRs), and the p5 promoter/ori driving expression of the AAV rep gene. A detailed understanding of the properties of these origins of DNA replication and the molecular mechanisms of interactions between them, may allow designing novel hybrid vectors that allow the efficient and precise integration of large transgenes in the human genome.     

Evaluation of risks related to the use of adeno-associated virus-based vectors

Recombinant AAV efficacy has been demonstrated in numerous gene therapy preclinical studies. As this vector is increasingly applied to human clinical trials, it is a priority to evaluate the risks of its use for workers involved in research and clinical trials as well as for the patients and their descendants. At high multiplicity of infection, wild-type AAV integrates into human chromosome 19 in approximately 60% of latently infected cell lines. However, it has been recently demonstrated that only approximately 1 out of 1000 infectious units can integrate. The mechanism of this site-specific integration involves AAV Rep proteins which are absent in vectors. Accordingly, recombinant AAV (rAAV) do not integrate site-specifically. Random integration of vector sequences has been demonstrated in established cell lines but only in some cases and at low frequency in primary cultures and in vivo. In contrast, episomal concatemers predominate.Therefore, the risks of insertional mutagenesis and activation of oncogenes are considered low. Biodistribution studies in non-human primates after intramuscular, intrabronchial, hepatic artery and subretinal administration showed low and transient levels of vector DNA in body fluids and distal organs. Analysis of patients body fluids revealed rAAV sequences in urine, saliva and serum at short-term. Transient shedding into the semen has been observed after delivery to the hepatic artery. However, motile germ cells seemed refractory to rAAV infection even when directly exposed to the viral particles, suggesting that the risk of insertion of new genetic material into the germ line is absent or extremely low. Risks related to viral capsid-induced inflammation also seem to be absent since immune response is restricted to generation of antibodies. In contrast, transgene products can elicit both cellular and humoral immune responses, depending on the nature of the expressed protein and of the route of vector administration. Finally, a correlation between early abortion as well as male infertility and the presence of wt AAV DNA in the genital tract has been suggested. Although no causal relationship has been established, this issue stresses the importance of using rAAV stocks devoid of contaminating replication-competent AAV. This review comprehensively examines virus integration, biodistribution, immune interactions, and other safety concerns regarding the wild-type AAV and recombinant AAV vectors.     

Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.     

Gene therapy vectors based on adeno-associated virus: characteristics and applications to acquired and inherited diseases (review)

Adeno-associated virus (AAV), a defective parvovirus, was discovered more than 30 years ago. Interest in this virus for human gene therapy applications focuses on its non-pathogenicity, broad tropism and infectivity, site-specific integration and long-term persistence. The field of rAAV research has considerably advanced: titers of 1014 p/ml have been achieved, plasmid systems devised to produce helper-free viruses, chimaeric vectors combining properties of rAAV ITRs and large sequence capacity from Ad/HS vectors in parallel with the revolutionary intron strategy based on heterodimerisation of the forming concatamers have expanded the vector capacity. Muscle cells and neurons (post-mitotic cells) are amongst the most efficient targets of rAAV delivery and AAV receptors and co-receptors have been identified. This review will describe advances in the field of rAAV technology that overcome certain limitations of the vector as a gene delivery system and overview applications involving these recombinant vectors for the treatment of acquired and inherited diseases.     

Adeno-associated virus vectors for human gene therapy

Adeno-associated virus(AAV) is a small,non-enveloped virus that contains a single-stranded DNA genome. It was the first gene therapy drug approved in the Western world in November 2012 to treat patients with lipoprotein lipase deficiency. AAV made history and put human gene therapy in the forefront again. More than four decades of research on AAV vector biology and human gene therapy has generated a huge amount of valuable information. Over 100 AAV serotypes and variants have been isolated and at least partially characterized. A number of them have been used for preclinical studies in a variety of animal models. Several AAV vector production platforms,especially the baculovirus-based system have been established for commercial-scale AAV vector production. AAV purification technologies such as density gradient centrifugation,column chromatography,or a combination,have been well developed. More than 117 clinical trials have been conducted with AAV vectors. Although there are still challenges down the road,such as crossspecies variation in vector tissue tropism and gene transfer efficiency,pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients,the gene therapy community is forging ahead with cautious optimism. In this review I will focus on the properties and applications of commonly used AAV serotypes and variants,and the technologies for AAV vector production and purification. I will also discuss the advancement of several promising gene therapy clinical trials.     

New recombinant serotypes 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.     

Immune responses to adeno-associated virus vectors

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.     

Synergistic inhibition of PARP‐1 and NF‐κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy

Host immune response remains a key obstacle to widespread application of adeno‐associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP‐ribose) polymerase‐1 (PARP‐1) negatively affect the integration of AAV in the host genome. However, the role of PARP‐1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP‐1 improves the transduction of single‐stranded AAV vectors both in vitro (～174%) and in vivo (two‐ to 3.4‐fold). Inhibition of PARP‐1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF‐κB subunit proteins associated with the host innate response against self‐complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP‐1 and NF‐κB signaling. This strategy also effectively attenuated the AAV capsid‐specific cytotoxic T‐cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV‐mediated gene therapy.     

Natural infection with adeno-associated viruses

Adeno-associated viruses (AAV) are parvoviruses which exhibit oncosuppressive properties as well as unique characteristics of integration. They have never been associated with human diseases. AAV are thus considered promising vectors for the corrective therapy of various gene defects. In this review, the (possible) consequences of AAV infection for human health, cancer development and recombinant AAV vectors are discussed with respect to recent results on the cellular and molecular targets of AAV infection in humans.     

Adeno-Associated Virus (AAV) Vectors in the CNS

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, as well as the creation of novel chimeric 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. In many cases the proposed therapies have progressed to phase I/II clinical trials. Each individual application, however, presents a unique set of challenges that must be solved in order to attain clinically effective gene therapies.     

Adeno-associated virus (AAV) vectors in the CNS

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.     

Adeno‐associated virus (AAV) vectors in gene therapy: immune challenges and strategies to circumvent them

AAV‐based gene transfer protocols have shown remarkable success when directed to immune‐privileged sites such as for retinal disorders like Lebers congenital amaurosis. In contrast, AAV‐mediated gene transfer into liver or muscle tissue for diseases such as hemophilia B, α1 anti‐trypsin deficiency and muscular dystrophy has demonstrated a decline in gene transfer efficacy over time. It is now known that in humans, AAV triggers specific pathways that recruit immune sensors. These factors initiate an immediate reaction against either the viral capsid or the vector encoded protein as part of innate immune response or to produce a more specific adaptive response that generates immunological memory. The vector‐transduced cells are then rapidly destroyed due to this immune activation. However, unlike other viral vectors, AAV is not immunogenic in murine models. Its immunogenicity becomes apparent only in large animal models and human subjects. Moreover, humans are natural hosts to AAV and exhibit a high seroprevalence against AAV vectors. This limits the widespread application of AAV vectors into patients with pre‐existing neutralising antibodies or memory T cells. To address these issues, various strategies are being tested. Alternate serotype vectors (AAV1‐10), efficient expression cassettes, specific tissue targeting, immune‐suppression and engineered capsid variants are some approaches proposed to minimise this immune stimulation. In this review, we have summarised the nature of the immune response documented against AAV in various pre‐clinical and clinical settings and have further discussed the strategies to evade them. Copyright © 2013 John Wiley & Sons, Ltd.     

From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy

Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of "pseudotyped" AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.     

Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy in monkey

Gene therapy is emerging as a therapeutic modality for treating disorders of the retina. Photoreceptor cells are the primary cell type affected in many inherited diseases of retinal degeneration. Successfully treating these diseases with gene therapy requires the identification of efficient and safe targeting vectors that can transduce photoreceptor cells. One serotype of adeno-associated virus, AAV2, has been used successfully in clinical trials to treat a form of congenital blindness that requires transduction of the supporting cells of the retina in the retinal pigment epithelium (RPE). Here, we determined the dose required to achieve targeting of AAV2 and AAV8 vectors to photoreceptors in nonhuman primates. Transgene expression in animals injected subretinally with various doses of AAV2 or AAV8 vectors carrying a green fluorescent protein transgene was correlated with surgical, clinical, and immunological observations. Both AAV2 and AAV8 demonstrated efficient transduction of RPE, but AAV8 was markedly better at targeting photoreceptor cells. These preclinical results provide guidance for optimal vector and dose selection in future human gene therapy trials to treat retinal diseases caused by loss of photoreceptors.     

Immune responses to AAV in clinical trials

Findings in the first clinical trial in which an adeno-associated virus (AAV) vector was introduced into the liver of human subjects highlighted an issue not previously identified in animal studies. Upon AAV gene transfer to liver, two subjects developed transient elevation of liver enzymes, likely as a consequence of immune rejection of transduced hepatocytes mediated by AAV capsid-specific CD8(+) T cells. Studies in healthy donors showed that humans carry a population of antigen-specific memory CD8(+) T cells probably arising from wild-type AAV infections. The hypothesis formulated at that time was that these cells expanded upon re-exposure to capsid, i.e. upon AAV-2 hepatic gene transfer, and cleared AAV epitope-bearing transduced hepatocytes. Other hypotheses have been formulated which include specific receptor-binding properties of AAV-2 capsid, presence of capsid-expressing DNA in AAV vector preparations, and expression of alternate open reading frames from the transgene; emerging data from clinical trials however fail to support these competing hypotheses. Possible solutions to the problem are discussed, including the administration of a short-term immunosuppression regimen concomitant with gene transfer, or the development of more efficient vectors that can be administered at lower doses. While more studies will be necessary to define mechanisms and risks associated with capsid-specific immune responses in humans, monitoring of these responses in clinical trials will be essential to achieving the goal of long-term therapeutic gene transfer in humans.     

Diverse IgG subclass responses to adeno‐associated virus infection and vector administration

Humoral immune responses occur following exposure to Adeno‐associated virus (AAV) or AAV vectors. Many studies characterized antibody responses to AAV, but human IgG subclass responses to AAV have not been previously described. In this study, IgG subclass responses were examined in serum samples of normal human subjects exposed to wild‐type AAV, subjects injected intramuscularly with AAV vectors and subjects injected intravascularly with AAV vectors. A diversity of IgG subclass responses to AAV capsid were found in different subjects. IgG1 was found to be the dominant response. IgG2, IgG3, and IgG4 responses were also observed in most normal human subjects; IgG2 and IgG3 each represented the major fraction of total anti‐AAV capsid IgG in a subset of normal donors. Subjects exposed to AAV vectors showed IgG responses to AAV capsid of all four IgG subclasses. IgG responses to AAV capsid in clinical trial subjects were inversely proportional to the level of pre‐existing anti‐AAV antibody and independent of the vector dose. The high levels of anti‐AAV capsid IgG1 can mask differences in IgG2, IgG3, and IgG4 responses that were observed in this study. Analysis of IgG subclass distribution of anti‐AAV capsid antibodies indicates a complex, non‐uniform pattern of responses to this viral antigen. J. Med. Virol. 81:65–74, 2009. © 2008 Wiley‐Liss, Inc.     

Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model.

Recombinant vectors based on adeno-associated virus (AAV) or human immunodeficiency 1 (lentivirus) are promising tools for long term in vivo gene delivery. Their design allows the exchange of capsids or envelopes, respectively, theoretically providing the opportunity to transduce a range of cell types. We constructed AAV vectors encoding enhanced green fluorescent protein (EGFP) within an AAV serotype 2 (AAV2) genome contained in an AAV2, five or one capsid (called AAV2/2, AAV2/5 and AAV2/1, respectively). Similarly we produced lentiviral vectors, encoding the same expression cassette present in the AAV vectors, pseudotyped with proteins from vesicular stomatitis virus glycoprotein (VSVG) or Mokola envelopes. Transduction characteristics of these vectors were evaluated in the murine retina following subretinal or intravitreal administration. The time of onset of transgene expression and the targeted cell types differed between the various recombinants. Onset of transgene expression was 3-4 days for lentiviral vectors and AAV2/1. In contrast, onset was at 2-4 weeks for AAV2/5 and AAV2/2, respectively. After subretinal injection, both lenti-VSVG and AAV2/5 transduced the retinal pigment epithelium (RPE) and photoreceptors efficiently whereas transgene expression was restricted to RPE cells using lenti with the Mokola envelope or AAV2/1. After intravitreal administration, only AAV2/2 and lenti-VSVG transduced the inner retina. Vector-mediated fluorescence was detected in the retina for over 12 weeks for all of the vectors. We conclude that pseudotyping provides a useful means to manipulate viral vector cell targeting specificity as well as retinal transduction characteristics of vectors containing the same genome.