Regulation of gene expression in adeno-associated virus vectors in the brain

Regulated adeno-associated virus (AAV) vectors have broad utility in both experimental and applied gene therapy, and to date, several regulation systems have exhibited a capability to control gene expression from viral vectors over two orders of magnitude. The tetracycline responsive system has been the most used in AAV, although other regulation systems such as RU486- and rapamycin-responsive systems are reasonable options. AAV vectors influence how regulation systems function by several mechanisms, leading to increased background gene expression and restricted induction. Methods to reduce background expression continue to be explored and systems not yet tried in AAV may prove quite functional. Although regulated promoters are often assumed to exhibit ubiquitous expression, the tropism of different neuronal subtypes can be altered dramatically by changing promoters in recombinant AAV vectors. Differences in promoter-directed tropism have significant consequences for proper expression of gene products as well as the utility of dual vector regulation. Thus regulated vector systems must be carefully optimized for each application.     

Proteasome inhibition is partially effective in attenuating pre-existing immunity against recombinant adeno-associated viral vectors

Pre-existing immunity against adeno-associated virus (AAV) remains a major challenge facing the clinical use of systemic administration of recombinant AAV vectors for the treatment of genetic and acquired diseases using gene therapy. In this study, we evaluated the potential of bortezomib (marketed under trade name Velcade) to abrogate a pre-existing immunity to AAV in mice, thereby allowing subsequent transduction by a recombinant AAV vector of the same serotype. We demonstrate that bortezomib efficiently reduces AAV-specific IgG titres and moderates the cytotoxic T cell response in mice that have a pre-existing immunity to AAV2/8. Significant depletion of AAV2/8-specific IgG-producing plasma cells in secondary lymphoid organs and bone marrow was observed. However, this inhibition of the immune response by bortezomib was insufficient to allow subsequent re-infection with a recombinant AAV vector of a similar serotype. We show that this shortcoming is probably due to the combination of residual antibody levels and the inability of bortezomib to completely deplete the memory B cells that are re-activated in response to a repeated infection with a recombinant AAV vector. Taken together, the results of this study argue for the use of immunosuppressive therapies that target both plasma and memory B cells for the efficient elimination of pre-existing immunity against AAV2/8 vectors.     

Adeno-associated viral vectors for gene transfer and gene therapy.

Adeno-associated virus (AAV) is a defective, non-pathogenic human parvovirus that depends for growth on coinfection with a helper adenovirus or herpes virus. Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as vectors for gene therapy. In contrast to other gene delivery systems, rAAVs lack all viral genes and show long-term gene expression in vivo without immune response or toxicity. Over the past few years, many applications of rAAVs as therapeutic agents have demonstrated the utility of this vector system for long-lasting genetic modification and gene therapy in preclinical models of human disease. New production methods have increased rAAV vector titers and eliminated contamination by adenovirus. In addition, vectors for regulatable gene expression and vectors retargeted to different cells have been engineered. These advancements are expected to accelerate and facilitate further animal model studies, providing validation for use of rAAVs in human clinical trials.     

Site-specific integration of a transgene mediated by a hybrid adenovirus/adeno-associated virus vector using the Cre/loxP-expression-switching system

As vectors, adenoviruses (Ads) have many attractive advantages for in vivo gene therapy. However, Ads do not usually integrate into the host genome and gene expression is, thus, transient. Adeno-associated virus (AAV) integrates into a specific locus (AAVS1) on the human host's chromosome 19, while conventional recombinant AAV (rAAV) vectors do not possess this property because such vectors lack the rep gene. AAV vectors carrying the rep gene do not have enough space for insertion of a transgene. We have constructed a hybrid adenovirus/adeno-associated virus (Ad/AAV) vector which has the advantages of both Ads and AAVs. Given that the rep gene products inhibit propagation of Ads, we used the Cre/loxP-expression-switching system to regulate the expression of the rep gene. The Ad/AAV vector easily propagates, can efficiently infect a broad range of cell types, and can integrate into a specific locus on host chromosomes.     

Adeno-associated virus vector-mediated gene delivery to the vasculature and kidney

Relatively successful elsewhere, gene delivery aimed at the vasculature and kidney has made very little progress. In the kidney, the hurdles are related to the unique structure-function relationships of this organ and in the blood vessels to a variety of, mostly endothelial, factors making the delivery of transgenes very difficult. Among gene-therapeutic approaches, most viral gene delivery systems utilized to date have shown significant practical and safety-related limitations due to the level and duration of recombinant transgene expression as well as their induction of a significant host immune response to vector proteins. Recombinant adeno-associated virus (rAAV) vectors appear to offer a vehicle for safe, long-term transgene expression. rAAV-based vectors are characterized by a relative non-immunogenicity and the absence of viral coding sequences. Furthermore, they allow for establishment of long-term latency without deleterious effects on the host cell. This brief review addresses problems related to transgene-delivery to kidney and vasculature with particular attention given to rAAV vectors. The potential for gene therapy as a strategy for selected renal and vascular diseases is also discussed.     

Transgene Regulation Using the Tetracycline-Inducible TetR-KRAB System after AAV-Mediated Gene Transfer in Rodents and Nonhuman Primates

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.     

Recent developments in adeno-associated virus vector technology

Adeno-associated virus (AAV), a single-stranded DNA parvovirus, is emerging as one of the leading gene therapy vectors owing to its nonpathogenicity and low immunogenicity, stability and the potential to integrate site-specifically without known side-effects. A portfolio of recombinant AAV vector types has been developed with the aim of optimizing efficiency, specificity and thereby also the safety of in vitro and in vivo gene transfer. More and more information is now becoming available about the mechanism of AAV/host cell interaction improving the efficacy of recombinant AAV vector (rAAV) mediated gene delivery. This review summarizes the current knowledge of the infectious biology of AAV, provides an overview of the latest developments in the field of AAV vector technology and discusses remaining challenges.     

Efficient generation and amplification of high-capacity adeno-associated virus/adenovirus hybrid vectors

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.     

High-titer, wild-type free recombinant adeno-associated virus vector production using intron-containing helper plasmids

Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate the rep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.     

Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562–566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111–1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and acquired human diseases affecting cells of erythroid lineage.     

Comparative Analysis of DNA Nanoparticles and AAVs for Ocular Gene Delivery

Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV), makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs), critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg) than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.     

Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration.

The ability of recombinant adeno-associated virus (AAV) vectors to integrate into the host genome and to transduce nondividing cells makes them attractive as vehicles for gene delivery. In this study, we assessed the ability of several AAV vectors to transduce airway cells in rabbits by measuring marker gene expression. AAV vectors that transferred either a beta-galactosidase (beta-gal) or a human placental alkaline phosphatase (AP) gene were delivered to one lobe of the rabbit lung by use of a balloon catheter placed under fluoroscopic guidance. We observed vector-encoded beta-gal or AP staining almost exclusively in the epithelial and smooth muscle cells in the bronchus at the region of balloon placement. The overall efficiency of transduction in the balloon-treated bronchial epithelium was low but reached 20% in some areas. The majority of the staining was in ciliated cells but was also observed in basal cells and airway smooth muscle cells. We observed an 80-fold decrease in marker-positive epithelial cells during the 60-day period after vector infusion, whereas the number of marker-positive smooth muscle cells stayed constant. Although treatment with the topoisomerase inhibitor etoposide dramatically enhanced AAV transduction in primary airway epithelial cells in culture, treatment of rabbits did not improve transduction rates in the airway. Vector readministration failed to produce additional transduction events, which correlated with the appearance of neutralizing antibodies. These results indicate that both readministration and immune modulation will be required in the use of AAV vectors for gene therapy to the airway epithelium.     

Role for highly regulated rep gene expression in adeno-associated virus vector production.

Recent success achieving long-term in vivo gene transfer without a significant immune response by using adeno-associated virus (AAV) vectors (X. Xiao, J. Li, and R. J. Samulski, J. Virol. 70:8098-8108, 1996) has encouraged further development of this vector for human gene therapy. Currently, studies focus on the generation of high-titer vectors by using the two-plasmid helper-vector system in adenovirus (Ad)-infected cells. To examine the effects of the AAV replication (rep) genes on recombinant AAV (rAAV) vector production, we have constructed a series of AAV helper plasmids that contain strong heterologous promoters in place of the endogenous p5 promoter. Although high-level rep gene expression was achieved, rAAV DNA failed to replicate in the absence of Ad infection. Moreover, unregulated overexpression of Rep78/68 led to substantially lower rAAV yields in the presence of Ad (10(4-5) versus 10(7-8)). In contrast, under similar conditions, reduced Rep78/68 expression resulted in much higher rAAV yields (10(9)). Molecular characterization showed that overexpression of the rep gene decreased rAAV DNA replication and severely inhibited capsid (cap) gene expression. Interestingly, a reduced rep level enhanced cap gene expression and supported normal rAAV DNA replication. These studies suggest a critical role for regulated rep gene expression in rAAV production and have facilitated the development of a new AAV helper plasmid that increases vector production eightfold over currently used constructs.     

Induction of robust immune responses against human immunodeficiency virus is supported by the inherent tropism of adeno-associated virus type 5 for dendritic cells

The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.     

Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.     

Novel AAV serotypes for improved ocular gene transfer

Some of the most successful gene therapy results have been obtained using recombinant viral vectors to treat animal models of inherited and acquired ocular diseases. Clinical trials using adenovirus vector systems have been initiated for two ocular diseases. Adeno-associated viruses (AAVs) represent an attractive alternative to adenoviral vector systems as they enable stable and long-term expression and can target a variety of different ocular cell types depending on the capsid serotype; recently clinical trails for congenital blindness was initiated with a vector-based AAV serotype 2. High levels of retinal gene transfer have been achieved using vectors based on AAV serotypes 1, 2, 4 and 5. This report compares the gene transfer efficacy and stability of expression of vector systems based on three novel AAV serotypes: AAV7, 8, 9, with the established vectors AAV1, 2, 5. We show here that AAV7 and 8 enable superior long-term transduction of retinal and also anterior chamber structures.     

Repeated Delivery of Adeno-Associated Virus Vectors to the Rabbit Airway

Efficient local expression from recombinant adeno-associated virus (rAAV)-cystic fibrosis (CF) transmembrane conductance regulator (CFTR) vectors has been observed in the airways of rabbits and monkeys for up to 6 months following a single bronchoscopic delivery. However, it is likely that repeated administrations of rAAV vectors will be necessary for sustained correction of the CF defect in the airways. The current study was designed to test the feasibility of repeated airway delivery of rAAV vectors in the rabbit lung. After two doses of rAAV-CFTR to the airways, rabbits generated high titers of serum anti-AAV neutralizing antibodies. Rabbits then received a third dose of a rAAV vector containing the green fluorescent protein (GFP) reporter gene packaged in either AAV serotype 2 (AAV2) or serotype 3 (AAV3) capsids. Each dose consisted of 1 ml containing 5 x 10(9) DNase-resistant particles of rAAV vector, having no detectable replication-competent AAV or adenovirus. Three weeks later, GFP expression was observed in airway epithelial cells despite high anti-AAV neutralizing titers at the time of delivery. There was no significant difference in the efficiency of DNA transfer or expression between the rAAV3 and rAAV2 groups. No significant inflammatory responses to either repeated airway exposure to rAAV2-CFTR vectors or to GFP expression were observed. These experiments demonstrate that serum anti-AAV neutralizing antibody titers do not predict airway neutralization in vivo and that repeated airway delivery rAAV allows for safe and effective gene transfer.