Ability Of Adeno-associated Virus Research Articles

Adeno-Associated virus 8 delivers an immunomodulatory peptide to mouse liver more efficiently than to rat liver.

Targeting the Kv1.3 potassium channel has proven effective in reducing obesity and the severity of animal models of autoimmune disease. Stichodactyla toxin (ShK), isolated from the sea anemone Stichodactyla helianthus, is a potent blocker of Kv1.3. Several of its analogs are some of the most potent and selective blockers of this channel. However, like most biologics, ShK and its analogs require injections for their delivery, and repeated injections reduce patient compliance during the treatment of chronic diseases. We hypothesized that inducing the expression of an ShK analog by hepatocytes would remove the requirement for frequent injections and lead to a sustained level of Kv1.3 blocker in the circulation. To this goal, we tested the ability of Adeno-Associated Virus (AAV)8 vectors to target hepatocytes for expressing the ShK analog, ShK-235 (AAV-ShK-235) in rodents. We designed AAV8 vectors expressing the target transgene, ShK-235, or Enhanced Green fluorescent protein (EGFP). Transduction of mouse livers led to the production of sufficient levels of functional ShK-235 in the serum from AAV-ShK-235 single-injected mice to block Kv1.3 channels. However, AAV-ShK-235 therapy was not effective in reducing high-fat diet-induced obesity in mice. In addition, injection of even high doses of AAV8-ShK-235 to rats resulted in a very low liver transduction efficiency and failed to reduce inflammation in a well-established rat model of delayed-type hypersensitivity. In conclusion, the AAV8-based delivery of ShK-235 was highly effective in inducing the secretion of functional Kv1.3-blocking peptide in mouse, but not rat, hepatocytes yet did not reduce obesity in mice fed a high-fat diet.

Comparison of Gene Delivery to the Kidney by Adenovirus, Adeno-Associated Virus, and Lentiviral Vectors After Intravenous and Direct Kidney Injections.

There are many kidney diseases that might be addressed by gene therapy. However, gene delivery to kidney cells is inefficient. This is due, in part, to the fact that the kidney excludes molecules above 50 kDa and that most gene delivery vectors are megaDaltons in mass. We compared the ability of adeno-associated virus (AAV), adenovirus (Ad), and lentiviral (LV) vectors to deliver genes to renal cells. When vectors were delivered by the intravenous (IV) route in mice, weak luciferase activity was observed in the kidney with substantially more in the liver. When gene delivery was observed in the kidney, expression was primarily in the glomerulus. To avoid these limitations, vectors were injected directly into the kidney by retrograde ureteral (RU) and subcapsular (SC) injections in mice. Small AAV vectors transduced the kidney, but also leaked from the organ and mediated higher levels of transduction in off-target tissues. Comparison of AAV2, 6.2, 8, and rh10 vectors by direct kidney injection demonstrated highest delivery by AAV6.2 and 8. Larger Ad and LV vectors transduced kidney cells and mediated less off-target tissue transduction. These data demonstrate the utility of direct kidney injections to circumvent the kidney size exclusion barrier. They also identify the effects of vector size on on-target and off-target transduction. This lays the foundation for the use of different vector platforms for gene therapy of diverse kidney diseases.

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose–response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2–500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8+ T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX–/– mice 8–10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100–500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).

Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

AAV-mediated gene transfer of human pigment epithelium-derived factor inhibits Lewis lung carcinoma growth in mice

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, and mechanisms through which PEDF exerts its antitumour activity have recently been defined. The aim of our research was to evaluate the ability of adeno-associated virus (AAV) vector-mediated transfer of human PEDF to inhibit Lewis lung carcinoma (LCC) cell growth. Intratumoural injection of AAV-PEDF caused significant reduction of the tumour volume and prolonged the survival time of mice bearing LLC cells, which were associated with decreased microvessel density and increased apoptosis in the tumours. AAV vectors represent a very promising tool for cancer gene therapy. No noticeable toxicity concerning AAV was detected as inferred from monitoring changes in animal body weight as well as basic organ structure and histological morphology, and by analyzing mouse liver and kidney function. Our findings indicate that AAV-mediated PEDF gene expression may offer an active approach to inhibit LLC growth and that treatment with AAV-PEDF may provide a promising therapeutic strategy in lung cancer treatment.

Liver Production of Sulfamidase Reverses Peripheral and Ameliorates CNS Pathology in Mucopolysaccharidosis IIIA Mice

Mucopolysaccharidosis type IIIA (MPSIIIA) is an inherited lysosomal storage disease caused by deficiency of sulfamidase, resulting in accumulation of the glycosaminoglycan (GAG) heparan sulfate. It is characterized by severe progressive neurodegeneration, together with somatic alterations, which lead to death during adolescence. Here, we tested the ability of adeno-associated virus (AAV) vector-mediated genetic modification of either skeletal muscle or liver to revert the already established disease phenotype of 2-month-old MPSIIIA males and females. Intramuscular administration of AAV-Sulfamidase failed to achieve significant therapeutic benefit in either gender. In contrast, AAV8-mediated liver-directed gene transfer achieved high and sustained levels of circulating active sulfamidase, which reached normal levels in females and was fourfold higher in males, and completely corrected lysosomal GAG accumulation in most somatic tissues. Remarkably, a 50% reduction of GAG accumulation was achieved throughout the entire brain of males, which correlated with a partial improvement of the pathology of cerebellum and cortex. Liver-directed gene transfer expanded the lifespan of MPSIIIA males, underscoring the importance of reaching supraphysiological plasma levels of enzyme for maximal therapeutic benefit. These results show how liver-directed gene transfer can reverse somatic and ameliorate neurological pathology in MPSIIIA.

AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers

In liver cirrhosis, abnormal liver architecture impairs efficient transduction of hepatocytes with large viral vectors such as adenoviruses. Here we evaluated the ability of adeno-associated virus (AAV) vectors, small viral vectors, to transduce normal and cirrhotic rat livers. Using AAV serotype-1 (AAV1) encoding luciferase (AAV1Luc) we analyzed luciferase expression with a CCD camera. AAV1Luc was injected through the hepatic artery (intra-arterial (IA)), the portal vein (intra-portal (IP)), directly into the liver (intra-hepatic (IH)) or infused into the biliary tree (intra-biliar). We found that AAV1Luc allows long-term and constant luciferase expression in rat livers. Interestingly, IP administration leads to higher expression levels in healthy than in cirrhotic livers, whereas the opposite occurs when using IA injection. IH administration leads to similar transgene expression in cirrhotic and healthy rats, whereas intra-biliar infusion is the least effective route. After 70% partial hepatectomy, luciferase expression decreased in the regenerating liver, suggesting lack of efficient integration of AAV1 DNA into the host genome. AAV1Luc transduced mainly the liver but also the testes and spleen. Within the liver, transgene expression was found mainly in hepatocytes. Using a liver-specific promoter, transgene expression was detected in hepatocytes but not in other organs. Our results indicate that AAVs are convenient vectors for the treatment of liver cirrhosis.

Dopaminergic pathway reconstruction by Akt/Rheb‐induced axon regeneration

A prevailing concept in neuroscience has been that the adult mammalian central nervous system is incapable of restorative axon regeneration. Recent evidence, however, has suggested that reactivation of intrinsic cellular programs regulated by protein kinase B (Akt)/mammalian target of rapamycin (mTor) signaling may restore this ability. To assess this possibility in the brain, we have examined the ability of adenoassociated virus (AAV)-mediated transduction of dopaminergic neurons of the substantia nigra (SN) with constitutively active forms of the kinase Akt and the GTPase Ras homolog enriched in brain (Rheb) to induce regrowth of axons after they have been destroyed by neurotoxin lesion. Both constitutively active myristoylated Akt and hRheb(S16H) induce regrowth of axons from dopaminergic neurons to their target, the striatum. Histological analysis demonstrates that these new axons achieve morphologically accurate reinnervation. In addition, functional reintegration into target circuitry is achieved, as indicated by partial behavioral recovery. We conclude that regrowth of axons within the adult nigrostriatal projection, a system that is prominently affected in Parkinson's disease, can be achieved by activation of Akt/mTor signaling in surviving endogenous mesencephalic dopaminergic neurons by viral vector transduction.

Transduction Efficiencies of Novel AAV Vectors in Mouse Airway Epithelium In Vivo and Human Ciliated Airway Epithelium In Vitro

We have characterized the ability of adeno-associated virus (AAV) serotypes 1-9 in addition to nineteen novel vectors isolated from various tissues, to transduce mouse and human ciliated airway epithelium (HAE). Vectors expressing alpha-1-antitrypsin (AAT) and beta-galactosidase were co-instilled into the mouse lung. Of all the vectors tested rh.64R1, AAV5 and AAV6 were the most efficient. The high transduction observed in mouse was reproduced in HAE cell cultures for both rh.64R1 and AAV6 but not for AAV5. Since AAV6 was the most efficient vector in mouse and HAE we also tested the transduction efficiencies of the AAV6 singleton vectors (i.e., AAV6 variants with targeted mutations) in these models. Of these, AAV6.2 transduced mouse airway epithelium and HAE with greater efficiency than all other AAV vectors tested. We demonstrated that AAV6.2 exhibits improved transduction efficiency compared to previously reported AAVs in mouse airways and in culture models of human airway epithelium and that this vector requires further development for preclinical and clinical testing.

Adeno-associated virus vector-mediated delivery of pigment epithelium-derived factor restricts neuroblastoma angiogenesis and growth

Purpose The purpose of this study was to evaluate the ability of adeno-associated virus (AAV) vector-mediated delivery of pigment epithelium-derived factor (PEDF) to inhibit neuroblastoma (NB) xenograft growth. Pigment epithelium-derived factor was chosen for this study because, in addition to being a potent inhibitor of angiogenesis, it is capable of inducing neuronal differentiation. Methods Cohorts of mice received either recombinant AAV encoding human PEDF (rAAV-hPEDF) at a range of doses or control vector via tail vein. Subsequent hPEDF expression was measured by enzyme-linked immunoassay. After 6 weeks, the mice were given human NB cells by retroperitoneal injection and then killed 5 weeks later. Tumor weight, microvessel density, tumor differentiation, apoptosis, and levels of intratumoral vascular endothelial growth factor (VEGF) expression were determined at that time. In subsequent cohorts of mice, AAV-mediated murine PEDF expression was tested against both human NB xenografts and murine tumors. Results In a series of in vitro studies, PEDF was shown to inhibit endothelial cell activation and to stimulate differentiation of NB cell lines. After tail vein injection of rAAV-hPEDF, stable transgene expression was generated and correlated with levels of vector administration. Human NB xenograft growth was restricted by hPEDF in a dose-dependent fashion. Intratumoral VEGF expression and microvessel density were decreased, and tumor cell apoptosis was increased in PEDF-treated mice. Conclusions Treatment with PEDF had a significant impact on NB growth in mice when delivered continuously using a gene therapy–mediated approach. The activity of PEDF appears to be mediated in part by inhibition of tumor-induced angiogenesis through down-regulation of tumor-elaborated VEGF, with subsequent intratumoral apoptosis. Furthermore, hPEDF was able to induce NB differentiation in vitro and in vivo. In addition, antitumor efficacy was seen when mouse PEDF was used to treat syngeneic murine tumors. In our murine models, gene therapy–mediated delivery of PEDF appears promising for the treatment of neuroblastoma.

Neonatal intramuscular injection with recombinant adeno-associated virus results in prolonged beta-glucuronidase expression in situ and correction of liver pathology in mucopolysaccharidosis type VII mice.

For many metabolic diseases, early correction of the inherited deficiency is required to prevent long-term sequelae. We examined the ability of adeno-associated virus (AAV) to mediate efficient gene transfer during the neonatal period in mice with the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII). Quadriceps of newborn MPS VII mice were injected with an AAV vector containing human beta-glucuronidase (GUSB) cDNA. High-level intramuscular GUSB expression was seen as early as 2 weeks of age, and persisted for at least 16 weeks with no reduction in activity. In addition, GUSB activity was detected in both liver and spleen at later time points. The level of GUSB activity resulted in a significant reduction in lysosomal storage in the liver and a minimal reduction in the spleen at 16 weeks. However, the temporal and spatial pattern of hepatic GUSB activity, coupled with the presence of GUSB cDNA in liver sections, suggests that hematogenous dissemination of virus at the time of injection led to gene transfer to hepatic cells. These results demonstrate that AAV vectors can successfully infect neonatal muscle and persist through the rapid growth phase following birth. However, GUSB secretion from an intramuscular source is inefficient, limiting the therapeutic efficacy of this approach.