Adeno-associated Virus Transgene Research Articles

Conditional Regulation of Gene Expression by Ligand-Induced Occlusion of a MicroRNA Target Sequence.

RNA switches that modulate gene expression with small molecules have a number of scientific and clinical applications. Here, we describe a novel class of small regulatory on switches based on the ability of a ligand-bound aptamer to promote stem formation between a microRNA target sequence (miR-T) and a complementary competing strand. Two on switch architectures employing this basic concept were evaluated, differing in the location of a tetracycline aptamer and the region of a miR-21 target sequence (miR-21-T) masked by its competing strand. Further optimizations of miR-21-T and its competing strand resulted in tetracycline-regulated on switches that induced luciferase expression by 19-fold in HeLa cells. A similar switch design based on miR-122-T afforded 7-fold regulation when placed in tandem, indicating that this approach can be extended to additional miR-T. Optimized on switches introduced into adeno-associated virus (AAV) vectors afforded 10-fold regulation of two antiviral proteins in AAV-transduced cells. Our data demonstrate that small-molecule-induced occlusion of a miR-T can be used to conditionally regulate gene expression in mammalian cells and suggest that regulatory switches built on this principle can be used to dose expression of an AAV transgene.

Effects of Cellular Methylation on Transgene Expression and Site-Specific Integration of Adeno-Associated Virus

DNA methylation is a major epigenetic event that affects not only cellular gene expression but that also has the potential to influence bacterial and viral DNA in their host-dependent functions. Adeno-associated virus (AAV) genome contains a high degree of CpG sequences capable of methylation in its terminal repeat sequences, which are the sole elements retained in AAV-based vectors used in gene therapy. The present study determined the influence of methylation status of the host cell on wild type (wt) AAV integration and recombinant (r) AAV transgene expression in HeLa cells. Results of the study indicated that hypo-methylation significantly enhanced both wtAAV chromosomal integration and transgene expression of rAAV. A direct influence of methylation on AAV integration was further confirmed by methylating the AAVS1 integration sites prior to viral infection with DNA trans-complementation assay. These results signify the importance of epigenetic status of target cells as one of the key factors in long-term transgene expression in AAV gene therapy.

678. Increasing AAV Vector Yield By Riboswitch-Mediated Attenuation of Toxic Transgene Effects in HEK-293 Producer Cells

Adeno-associated virus (AAV) vectors are among the most promising viral vector systems for human gene therapy and have also been used extensively to investigate gene function in preclinical research. Achievement of high-titer vector yields regardless of the packaged transgene hence is a key goal of process optimization. However, transgenes expressed by constitutive, ubiquitously active promoters (such as the CMV promoter) often impair producer cell performance due to cytotoxic, anti-proliferative or other unknown effects, resulting in low vector yield.In our study, we explored artificial aptazyme riboswitches as novel RNA-intrinsic tools to regulate vector transgene activity during AAV production in HEK-293 cells. To this end, we integrated the guanine-responsive GuaM8HDV aptazyme in the UTR of a standard AAV transgene cassette and explored switching behavior under AAV production conditions.Our results demonstrate that transgene expression can be efficiently attenuated by a single addition of guanine in a routine medium exchange step during AAV production, thereby decreasing producer cell performance-impairing transgene effects such as cytotoxicity. In a proof-of-concept study using transgenes with both known (e.g. pro-apoptotic) as well as unknown modes of producer cell impairment, AAV vector yields could be boosted up to 23-fold with the riboswitch approach as compared to conventional vectors. As expected, this effect worked independently of the AAV serotype/capsid variant used. Importantly, GuaM8HDV-harboring AAV vectors preserved functionality (i.e. the ability to express the transgene) in vivo, as demonstrated in a mouse model of AAV-TGFb1-induced pulmonary fibrosis. Contrary to other inducible systems for gene expression control, the riboswitch system presented here does not require expression of additional transcription factors, but works in a small-molecule regulatable, RNA-intrinsic manner. Moreover, the riboswitch sequence only occupies ≈100 base pairs of plasmid space – a particular advantage for AAV vectors. Finally, this approach should in principle be expandable to other viral vector systems produced in mammalian cell culture such as Adenoviral vectors.Thus, we propose riboswitches as novel tools to foster transgeneindependent, high-titer production of viral vectors in mammalian cell culture systems. Moreover, our study further supports exploration of this technology for other purposes such as studying transgene dynamics and controlling transgene expression as a safety module in next-generation gene therapy vectors.

AAV shuffles to the liver: commentary on Lisowski et al.

AAV shuffles to the liver: commentary on Lisowski et al.

Successful Regional Delivery and Long-term Expression of a Dystrophin Gene in Canine Muscular Dystrophy: A Preclinical Model for Human Therapies

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.

Hoechst increases adeno‐associated virus‐mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter

In airway epithelia, the kinetics of recombinant adeno-associated virus (AAV) transgene expression is slow. This has negative practical implications for research, as well as for translation into therapy. The DNA minor groove-binding agent Hoechst-33342 has been shown to enhance AAV transgene expression. In the present study, we investigated the mechanism of Hoechst-related augmentation of AAV-mediated transgene expression. We investigated the effect of Hoechst-33342 on HT1080, COS-7, mouse and human airway epithelia transduced with different AAV serotypes encoding enhanced green fluorescent protein (eGFP). We exposed cells to increasing concentrations of Hoechst-33342 at different time points. We evaluated the effect on second-strand DNA synthesis using AAV with a self-complementary genome. We also investigated the effect on expression from transfected plasmids with and without AAV2 inverted terminal repeats (ITRs). We found that Hoechst-33342 significantly accelerated AAV transgene expression for all serotypes tested. Hoechst-33342 only had an effect when the treatment was given during or after transduction, even 120 days post-transduction, suggesting an effect on transgene expression regulation. Hoechst-33342 increased transgene expression when cells were transduced with a self-complementary AAV with the cytomegalovirus promoter, although there was no effect on cells transduced with conventional single-stranded AAV encoding the Rous sarcoma virus promoter. Finally, Hoechst-33342 increases gene expression from transfected plasmids regardless of the presence of AAV2 ITRs. Hoechst dramatically augments and accelerates AAV-mediated transgene expression in airway epithelia without altering AAV-mediated gene transfer. Hoechst activation of the cytomegalovirus promoter is seen in plasmids, although it is drastically enhanced in the context of AAV.

Immunity and AAV-Mediated Gene Therapy for Muscular Dystrophies in Large Animal Models and Human Trials.

Adeno-associated viral (AAV) vector-mediated gene replacement for the treatment of muscular dystrophy represents a promising therapeutic strategy in modern medicine. One major obstacle in using AAV vectors for in vivo gene delivery is the development of host immune responses to the viral capsid protein and transgene products as evidenced in animal models and human trials for a range of genetic diseases. Here, we review immunity against AAV vector and transgene in the context of gene delivery specific to muscles for treating muscular dystrophies and non-muscle diseases in large animal models and human trials, factors that influence the intensity of the immune responses, and immune modulatory strategies to prevent unwanted immune responses and induce tolerance to the vector and therapeutic gene for a successful gene therapy.

Proteasome Inhibitors Enhance Gene Delivery by AAV Virus Vectors Expressing Large Genomes in Hemophilia Mouse and Dog Models: A Strategy for Broad Clinical Application

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

Cytotoxic T lymphocyte responses to transgene product, not adeno-associated viral capsid protein, limit transgene expression in mice.

The use of adeno-associated viral (AAV) vectors for gene replacement therapy is currently being explored in several clinical indications. However, reports have suggested that input capsid proteins from AAV-2 vector particles may result in the stimulation of cytotoxic T lymphocyte (CTL) responses that can result in a loss of transduced cells. To explore the impact of anti-AAV CTLs on AAV-mediated transgene expression, both immunocompetent C57BL=6 mice and B cell-deficient muMT mice were immunized against the AAV2 capsid protein (Cap) and were injected intravenously with an AAV-2 vector encoding alpha-galactosidase (alpha-Gal). C57BL=6 mice, which developed both CTL and neutralizing antibody responses against Cap, failed to show any detectable alpha-Gal expression. In contrast, serum alpha-Gal levels comparable to those of naive mice were observed in muMT mice despite the presence of robust CTL activity against Cap, indicating that preexisting Cap-specific CTLs did not have any effect on the magnitude and duration of transgene expression. The same strategy was used to assess the impact of CTLs against the alpha-Gal transgene product on AAV-mediated gene delivery and persistence of transgene expression. Preimmunization of muMT mice with an Ad=alpha-Gal vector induced a robust CTL response to alpha-Gal. When these mice were injected with AAV2=alpha-Gal vector, initial levels of alpha-Gal expression were reduced by more than 1 log and became undetectable by 2 weeks postinjection. Overall, our results indicate that CTLs against the transgene product as opposed to AAV capsid protein are more likely to interfere with AAV transgene expression.

Adeno-associated Virus of a Single-polarity DNA Genome Is Capable of Transduction In Vivo

The adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. AAV vectors with wild-type inverted terminal repeats package either the plus- or the minus-strand DNA genomes with equal frequency. By creating a series of deletions within the, we have developed a genetic approach that can generate an AAV vector that packages its single-stranded DNA genome predominantly in a single polarity (99.4%). This novel reagent efficiently transduced muscle, brain and liver in whole animals. The transduction efficiencies were similar to those of the control mixed-polarity vectors. Our results showed that reannealing of plus- and minus-strand DNA was not required for AAV-mediated transduction in vivo, supporting the hypothesis that second-strand DNA synthesis is a primary pathway in converting the single-stranded AAV genome into double-stranded forms. The availability of the single-polarity AAV vector would aid further studies on the mechanism of AAV transduction as well as the application of AAV vector for gene replacement therapy.

80. Pre-Existing Cytotoxic T-Lymphocyte (CTL) Responses to the AAV CAP Protein Do Not Interfere with AAV Transgene Expression in Mice

The use of adeno-associated viral (AAV) vectors as a delivery method for gene replacement strategies is currently being explored in several clinical indications. AAV vectors do not express any viral proteins suggesting that they represent a less immunogenic delivery system. However, recent data have suggested that administration of AAV vectors may actually result in the stimulation of T-cell responses that result in a loss of transgene expression and may limit the utility of these vectors. By using computer prediction algorithms, we identified several AAV peptides as putative MHC I binders. Immunization of mice with peptides predicted to have the highest binding affinities did not result in a detectable cytotoxic T lymphocyte (CTL) response. In addition, intravenous administration of increasing doses of AAV serotype 2 vector also failed to induce a measurable CTL response. In contrast, immunization with either a plasmid or an adenoviral vector encoding the full length AAV2 CAP protein, to mimic exposure to wild type AAV, generated a potent CTL response. A pre-existing CTL response is likely to be present in the human population and C57Bl/6 IgH6 knock-out (KO) mice were used to determine whether such CTLs could affect AAV transduction and subsequent transgene expression. When immunized with two doses of CAP plasmid, the IgH6 KO mice mounted a robust CTL response without the generation of AAV neutralizing antibodies. The mice were then injected with an AAV2 vector encoding the alpha-galactosidase (a-gal) protein. AAV transduction was unimpeded and the serum levels of a-gal protein as well as the longevity of transgene expression were comparable to those observed in mice pre-immunized with empty plasmid as a control. In contrast, wild-type C57Bl/6 mice pre-immunized with CAP plasmid, which developed both CTLs and neutralizing antibodies against AAV, did not show any evidence of a-gal expression after the injection of vector. Taken together, results of these experiments suggest that pre-existing AAV-specific CTLs may have little or no effect on AAV transduction or transgene expression and confirm that neutralizing antibodies significantly interfere with transduction by AAV vector.

250. Protection Against Huntington's Diease Progression: AAV-Mediated Delivery of Biotherapeutics

Top of pageAbstract Advances in the development of adeno-associated viral (AAV) vectors for in vivo gene therapy have shown AAV vectors can safely direct the continuous, targeted production of biotherapeutics. This is promising for the treatment of neurological disorders like Huntington's disease where there is a delayed but progressive degeneration of the caudate-putamen. We have therefore investigated whether AAV-mediated in vivo gene therapy can effectively be used to deliver biotherapeutics and support vulnerable striatal neurons in a rodent model of Huntington's disease. Our focus is on brain- derived neurotrophic factor (BDNF) as a potential biotherapeutic, a protein important for general maintenance and functional plasticity of the striatum. We constructed a recombinant AAV vector with cDNA encoding human BDNF under the control of the chicken beta actin/CMV enhancer (CBA) constitutive promoter. The AAV expression cassettes are flanked by inverted terminal repeats from AAV serotype-2 and packaged using caspid proteins from both AAV serotype-1 and -2. Following AAV packaging we performed a protein assay with E15 primary striatal cells to check functional BDNF activity. Cells were transduced with AAV-BDNF and cultured in vitro for 3 weeks to allow neuronal differentiation. We observed a significant increase in calbindin positive neurons following AAV-BDNF transduction (P < 0.04) compared to the AAV-Luciferase vector indicating an increase in BDNF expression inducing neuronal differentiation towards a GABAergic phenotype. In vivo gene therapy was performed in adult male Wistar rats by stereotaxically injecting either AAV-BDNF (n = 12), AAV-Luciferase (n = 6) or vehicle solution (n = 6) into the striatum. We observed extensive AAV transduction and transgene protein expression within the striatum as well as the substantia nigra and globus pallidus due to anterograde transport of the vector via axonal projections. Three weeks following gene delivery the rats received a unilateral striatal injection of the neurotoxin quinolinic acid (50nmol) to model Huntington's disease cell loss. Forelimb use and sensorimotor neglect was analysed post lesioning to quantify motor function impairments between groups. The extent of neuroprotection was quantified by stereological analysis of DARPP-32 positive striatal projection neurons. We observed that BDNF over-expression within the striatum and projection nuclei provides protection against the excitotoxic cell death correlating with an alleviation of sensorimotor impairments. This study highlights the potential role of BDNF as an important biotherapeutic molecule for treating Huntington's disease and the use of in vivo gene therapy to limit neurodegeneration. Supported by Auckland Medical Research Foundation. A.P. Kells is a NZ FoRST Scholarship recipient.

Cochlear function and transgene expression in the guinea pig cochlea, using adenovirus- and adeno-associated virus-directed gene transfer.

Development of a viral vector that can infect hair cells of the cochlea without producing viral-associated ototoxic effects is crucial for utilizing gene replacement therapy as a treatment for certain forms of hereditary deafness. In the present study, cochlear function was monitored using distortion-product otoacoustic emissions (DPOAEs) in guinea pigs that received infusions of either (E1(-), E3(-)) adenovirus, or adeno-associated virus (AAV), directly into the scala tympani. Replication-deficient (E1(-), E3(-)) adenovirus-directed gene transfer, using the cytomegalovirus (CMV) promoter, drove transgene expression to inner hair cells and pillar cells of the cochlea. AAV transduction was tested with several promoters, such as platelet-derived growth factor (PDGF), neuron-specific enolase (NSE), and elongation factor 1alpha (EF-1alpha) promoters; which drove transgene expression to cochlear blood vessels, nerve fibers, and certain spiral limbus cells, respectively. AAV transgene expression was visualized by green fluorescent protein immunostaining. Immunocytochemistry to heparan sulfate confirmed the absence of proteoglycans in guinea pig hair cells, indicating that the receptor for AAV was not present on these cells. However, the heparan sulfate proteoglycan expression pattern mimicked the AAV transduction pattern. An overall finding was that cochlear function was not altered throughout the infection period using AAV titers as high as 5 x 10(8) IP/infused cochlea. In contrast, cochlear function was severely compromised by 8 days postinfection with adenoviral titers of 5 x 10(8) PFU/infused cochlea, and outer hair cells were eliminated. Thus, cochlear hair cells are amenable to in vivo gene transfer using a replication-deficient (E1(-), E3(-)) adenovirus. However, replication-defective or gutted adenovirus vectors must be employed to overcome the ototoxic effects of (E1(-), E3(-)) adenovirus vectors.