An Adeno-associated Virus Serotype 8 Research Articles

Therapeutic AAV9-mediated Suppression of Mutant SOD1 Slows Disease Progression and Extends Survival in Models of Inherited ALS

Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials.

Rescue of amyotrophic lateral sclerosis phenotype in a mouse model by intravenous AAV9‐ADAR2 delivery to motor neurons

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease, and the lack of effective therapy results in inevitable death within a few years of onset. Failure of GluA2 RNA editing resulting from downregulation of the RNA-editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) occurs in the majority of ALS cases and causes the death of motor neurons via a Ca2+-permeable AMPA receptor-mediated mechanism. Here, we explored the possibility of gene therapy for ALS by upregulating ADAR2 in mouse motor neurons using an adeno-associated virus serotype 9 (AAV9) vector that provides gene delivery to a wide array of central neurons after peripheral administration. A single intravenous injection of AAV9-ADAR2 in conditional ADAR2 knockout mice (AR2), which comprise a mechanistic mouse model of sporadic ALS, caused expression of exogenous ADAR2 in the central neurons and effectively prevented progressive motor dysfunction. Notably, AAV9-ADAR2 rescued the motor neurons of AR2 mice from death by normalizing TDP-43 expression. This AAV9-mediated ADAR2 gene delivery may therefore enable the development of a gene therapy for ALS.

P.4.3 Intravenous infusion of AAV8–MTM1 prolongs life and ameliorates severe muscle pathology in mouse and dog models of X-linked myotubular myopathy

Mutations in the myotubularin gene (MTM1) result in X-linked myotubular myopathy (XLMTM), a fatal pediatric disease of skeletal muscle characterized by small myofibers with frequent central nuclei and abnormal mitochondrial accumulations. Patients with XLMTM typically present with severe hypotonia, muscle weakness and respiratory failure. Previous local studies in Mtm1-mutant mice demonstrated potential efficacy of gene therapy to treat the disease. Here we report long-term survival data in mice and dogs following intravenous delivery of an adeno-associated virus serotype 8 (AAV8) vector expressing myotubularin under the muscle-specific desmin promoter. XLMTM dogs (<i>n</i>=3) were treated at 8weeks of age with full-length canine MTM1 cDNA using an AAV8 vector. Affected XLMTM dogs were infused with AAV8–MTM1 (2.5×10 13 vg/kg), IV under high pressure into the saphenous vein while a tourniquet was applied around the upper thigh. While AAV8–MTM1 was administered by hindlimb infusion, effects were observed systemically in dogs. Mice were given intravenous AAV8–MTM1 by tail vein injection. In both murine and canine models, myotubularin transgene was expressed in all limb muscles and this translated into marked improvement of contractile force in mice and dogs. All mutant mice, including those injected at a late stage of the disease, survived until the end of the 6-month study. The lifespan of MTM1 mutant dogs that normally survive to 4months was prolonged beyond 1year in the first of three treated animals. Two additional treated dogs remain robust and continue to thrive beyond 8months-of-age. Our findings in these two species provide proof-of-principle that intravenous AAV-mediated myotubularin replacement can rescue the severe phenotype of both small and large animal models of myotubular myopathy.

Enhancing the Utility of Adeno-Associated Virus Gene Transfer through Inducible Tissue-Specific Expression

The ability to regulate both the timing and specificity of gene expression mediated by viral vectors will be important in maximizing its utility. We describe the development of an adeno-associated virus (AAV)-based vector with tissue-specific gene regulation, using the ARGENT dimerizer-inducible system. This two-vector system based on AAV serotype 9 consists of one vector encoding a combination of reporter genes from which expression is directed by a ubiquitous, inducible promoter and a second vector encoding transcription factor domains under the control of either a heart- or liver-specific promoter, which are activated with a small molecule. Administration of the vectors via either systemic or intrapericardial injection demonstrated that the vector system is capable of mediating gene expression that is tissue specific, regulatable, and reproducible over induction cycles. Somatic gene transfer in vivo is being considered in therapeutic applications, although its most substantial value will be in basic applications such as target validation and development of animal models.

Adeno-Associated Virus Serotype 8 Gene Therapy Leads to Significant Lowering of Plasma Cholesterol Levels in Humanized Mouse Models of Homozygous and Heterozygous Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is a life-threatening genetic disease caused by mutations in the gene encoding low-density lipoprotein receptor (LDLR). As a bridge to clinical trials, we generated a "humanized" mouse model lacking LDLR and apolipoprotein B (ApoB) mRNA editing catalytic polypeptide-1 (APOBEC-1) expression and expressing a human ApoB100 transgene in order to permit more authentic simulation of in vivo interactions between the clinical transgene product, human LDLR (hLDLR), and its endogenous ligand, human ApoB100. On a chow diet, the humanized LDLR-deficient mice have substantial hypercholesterolemia and a lipoprotein phenotype more closely resembling human homozygous FH (hoFH) than in previous mouse models of FH. On injection of an adeno-associated virus serotype 8 (AAV8) vector encoding the human LDLR cDNA, significant correction of hypercholesterolemia was realized at doses as low as 1.5 × 10(11) genome copies (GC)/kg. Given that some patients with heterozygous FH (heFH) cannot be adequately treated with current therapy, we then extended our studies to similarly "humanized" mice that were heterozygous for LDLR deficiency, and that have a lipoprotein phenotype resembling heterozygous FH. Injection of AAV8-hLDLR brought about significant reduction in total and LDL cholesterol at doses as low as 5 × 10(11) GC/kg. Collectively, these data demonstrate the safety and efficacy of the liver-specific AAV8-hLDLR vector in the treatment of humanized mice modeling both hoFH and heFH.

Adeno-associated Virus Serotype 8 (AAV8) Delivery of Recombinant A20 to Skeletal Muscle Reduces Pathological Activation of Nuclear Factor (NF)-κB in Muscle of mdx Mice

Duchenne muscular dystrophy (DMD) is a genetic muscle disease caused by the absence of a functional dystrophin protein. Lack of dystrophin protein disrupts the dystrophin-glycoprotein complex causing muscle membrane instability and degeneration. One of the secondary manifestations resulting from lack of functional dystrophin in muscle tissue is an increased level of cytokines that recruit inflammatory cells, leading to chronic upregulation of the nuclear factor (NF)-κB. Negative regulators of the classical NF-κB pathway improve muscle health in the mdx mouse model for DMD. We have previously shown in vitro that a negative regulator of the NF-κB pathway, A20, plays a role in muscle regeneration. Here, we show that overexpression of A20 by using a muscle-specific promoter delivered with an adeno-associated virus serotype 8 (AAV8) vector to the mdx mouse decreases activation of the NF-κB pathway in skeletal muscle. Recombinant A20 expression resulted in a reduction in number of fibers with centrally placed nuclei and a reduction in the number of T cells infiltrating muscle transduced with the AAV8-A20 vector. Taken together, we conclude that overexpression of A20 in mdx skeletal muscle provides improved muscle health by reduction of chronic inflammation and muscle degeneration. These results suggest A20 is a potential therapeutic target to ameliorate symptoms of DMD.

Long-Term Expression and Safety of Administration of AAVrh.10hCLN2 to the Brain of Rats and Nonhuman Primates for the Treatment of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal, lysosomal storage disorder caused by mutations in the CLN2 gene, results in a deficiency of tripeptidyl-peptidase I (TPP-I) activity in neurons. Our prior studies showed that delivery of the human CLN2 cDNA directly to the CNS, using an adeno-associated virus serotype 2 (AAV2) vector, is safe in children with LINCL. As a second-generation strategy, we have demonstrated that AAVrh.10hCLN2, a rhesus-derived AAV vector, mediates wide distribution of TPP-I through the CNS in a murine model. This study tests the hypothesis that direct administration of AAVrh.10hCLN2 to the CNS of rats and nonhuman primates at doses scalable to humans has an acceptable safety profile and mediates significant CLN2 expression in the CNS. A dose of 10(11) genome copies (GC) was administered bilaterally to the striatum of Sprague Dawley rats with sacrifice at 7 and 90 days with no significant impact except for mild vector-related histopathological changes at the site of vector administration. A dose of 1.8×10(12) GC of AAVrh.10hCLN2 was administered to the CNS of 8 African green monkeys. The vector-treated monkeys did not differ from controls in any safety parameter except for mild to moderate white matter edema and inflammation localized to the administration sites of the vector. There were no clinical sequelae to these localized findings. TPP-I activity was >2 SD over background in 31.7±8.1% of brain at 90 days. These findings establish the dose and safety profile for human clinical studies for the treatment of LINCL with AAVrh.10hCLN2.

AAVrh.10-Mediated Expression of an Anti-Cocaine Antibody Mediates Persistent Passive Immunization That Suppresses Cocaine-Induced Behavior

Cocaine addiction is a major problem affecting all societal and economic classes for which there is no effective therapy. We hypothesized an effective anti-cocaine vaccine could be developed by using an adeno-associated virus (AAV) gene transfer vector as the delivery vehicle to persistently express an anti-cocaine monoclonal antibody in vivo, which would sequester cocaine in the blood, preventing access to cognate receptors in the brain. To accomplish this, we constructed AAVrh.10antiCoc.Mab, an AAVrh.10 gene transfer vector expressing the heavy and light chains of the high affinity anti-cocaine monoclonal antibody GNC92H2. Intravenous administration of AAVrh.10antiCoc.Mab to mice mediated high, persistent serum levels of high-affinity, cocaine-specific antibodies that sequestered intravenously administered cocaine in the blood. With repeated intravenous cocaine challenge, naive mice exhibited hyperactivity, while the AAVrh.10antiCoc.Mab-vaccinated mice were completely resistant to the cocaine. These observations demonstrate a novel strategy for cocaine addiction by requiring only a single administration of an AAV vector mediating persistent, systemic anti-cocaine passive immunity.

Frontotemporal lobar degeneration-related proteins induce only subtle memory-related deficits when bilaterally overexpressed in the dorsal hippocampus

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disease that involves cognitive decline and dementia. To model the hippocampal neurodegeneration and memory-related behavioral impairment that occurs in FTLD and other tau and TDP-43 proteinopathy diseases, we used an adeno-associated virus serotype 9 (AAV9) vector to induce bilateral expression of either microtubule-associated protein tau or transactive response DNA binding protein 43kDa (TDP-43) in adult rat dorsal hippocampus. Human wild-type forms of tau or TDP-43 were expressed. The vectors/doses were designed for moderate expression levels within neurons. Rats were evaluated for acquisition and retention in the Morris water task over 12weeks after gene transfer. Neither vector altered acquisition performance compared to controls. In measurements of retention, there was impairment in the TDP-43 group. Histological examination revealed specific loss of dentate gyrus granule cells and concomitant gliosis proximal to the injection site in the TDP-43 group, with shrinkage of the dorsal hippocampus. Despite specific tau pathology, the tau gene transfer surprisingly did not cause obvious neuronal loss or behavioral impairment. The data demonstrate that TDP-43 produced mild behavioral impairment and hippocampal neurodegeneration in rats, whereas tau did not. The models could be of value for studying mechanisms of FTLD and other diseases with tau and TDP-43 pathology in the hippocampus including Alzheimer's disease, with relevance to early stage mild impairment.

Effective reduction of the interleukin-1β transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis

To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1β (IL-1β) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding changes in the expression patterns of several critical disease mediators. Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1β transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify invitro transcript changes of IL-1β and an additional nine genes following transduction with this targeting knockdown vector. To validate invitro findings, this AAV5 vector was injected into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were calculated using the comparative CT (2(-ΔΔCT)) method. Statistically significant decreases in IL-1β expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups invitro. Transcript levels of anabolic transforming growth factor-β (TGF-β) were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-α (TNF-α), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-γ (IFN-γ), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. Invivo application of this targeting knockdown vector resulted in a >50% reduction (P=0.0045) or >90% (P=0.0001) of the IL-1β transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. Successful reduction of the IL-1β transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification. Investigations to characterize additional gene expression changes influenced by targeting knockdown AAV5 vector-based diminution of the IL-1β transcript invivo are warranted.

Adeno-Associated Virus Antibody Profiles in Newborns, Children, and Adolescents

Neutralizing antibodies (NAb) to an adeno-associated virus (AAV) vector due to previous natural infection with wild-type AAV can significantly limit gene transfer. NAb titers to AAV serotype 2 (AAV2) and AAV8 in human subjects (0 to 18 years) were studied. NAb prevalence is moderate at birth, decreases markedly from 7 to 11 months, and then progressively increases through childhood and adolescence.

An Adeno-Associated Virus Vector Efficiently and Specifically Transduces Mouse Skeletal Muscle

Expression of a therapeutic gene in the skeletal muscle is a practical strategy to compensate a patients' insufficient circulating factor. Its clinical application requires a muscle-targeting vector capable of inducing a continuous high-level transgene expression. We modified an adeno-associated virus serotype 2 (AAV2) vector expressing luciferase from the mouse muscle creatine kinase gene promoter-enhancer (Ckm). First, AAVS1 insulator was inserted into the vector genome for transcriptional enhancement. This increased transduction of mouse quadriceps muscle by 11-fold at 4weeks after intramuscular injection. Second, two capsid modifications were combined (21F capsid): incorporation of a segment of AAV1 capsid to produce a hybrid capsid and substitution of a tyrosine with a phenylalanine. Use of 21F capsid increased muscle transduction further by 18-fold, resulting in 200-fold higher efficacy than that of the unmodified vector. Compared with a vector having human elongation factor 1α promoter which showed similar efficacy in the muscle, this vector having Ckm transduced non-muscle organs less efficiently after intravenous administration. The AAV2 vector composed of the modified genome and capsid provides a backbone to develop a clinical vector expressing a therapeutic gene in the muscle.

Qualitative Imaging of Adeno-Associated Virus Serotype 2–Human Aromatic L-Amino Acid Decarboxylase Gene Therapy in a Phase I Study for the Treatment of Parkinson Disease

Putaminal convection-enhanced delivery (CED) of an adeno-associated virus serotype 2 (AAV2) vector, containing the human aromatic L-amino acid decarboxylase (hAADC) gene for the treatment of Parkinson disease (PD), has completed a phase I clinical trial. To retrospectively analyze magnetic resonance imaging (MRI) and positron emission tomography (PET) data from the phase I trial, correlate those data with similar nonhuman primate (NHP) data, and present how such information may improve future PD gene therapy trials in preparation for the initiation of the phase II trial. Ten patients with PD had been treated with bilateral MRI-guided putaminal infusions of AAV2-hAADC. MRI and PET scans were obtained at baseline (before vector administration) and at various intervals after treatment. Three normal adult NHPs received similar infusions into the thalamus. Imaging studies for both groups are presented, as well as hAADC immunohistochemistry for the NHPs. Early post-CED MRI confirmed the stereotactic targeting accuracy and revealed T2 hyperintensity around the distal cannula tracts, best seen within 4 hours of surgery. Coregistration of post-CED MRI and PET scans revealed increased PET uptake at the sites of T2 hyperintensity. Similar T2 hyperintensities in NHP MRI correlated with hAADC immunohistochemistry. Our analysis confirms the correct targeting of the CED cannula tracts within the target human putamen. Coregistration of MRI and PET confirms colocalization of T2 hyperintensities and increased PET uptake around the distal cannula tracts. Because PET uptake closely correlates with hAADC transgene expression and NHP data confirm this relationship between T2 hyperintensity and hAADC immunohistochemistry, we believe that T2-weighted MRI allows visualization of a significant part of the distribution volume of the hAADC gene therapy. Recommendations for future protocols based on these data are presented.

Evaluation of Systemic Follistatin as an Adjuvant to Stimulate Muscle Repair and Improve Motor Function in Pompe Mice

Due to the lack of acid alpha-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.

Replacement Gene Therapy with a HumanRPGRIP1Sequence Slows Photoreceptor Degeneration in a Murine Model of Leber Congenital Amaurosis

RPGR-interacting protein-1 (RPGRIP1) is localized in the photoreceptor-connecting cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone photoreceptor function and improved photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect.

A Phase 2 Study to Evaluate the Safety and Immunogenicity of a Recombinant HIV Type 1 Vaccine Based on Adeno-Associated Virus

The recombinant vaccine, tgAAC09, based on an adeno-associated virus serotype 2 (AAV2) vector encoding HIV-1 subtype C Gag, protease, and part of reverse transcriptase, induced robust T cell and antibody responses in nonhuman primates. In a previous phase I study in 80 healthy HIV-seronegative European and Indian adults, the vaccine was generally safe, well tolerated, and modestly immunogenic when administered once at doses up to 3 x 10(11) DRP. This phase II double-blind, randomized, placebo-controlled trial tested two administrations and a higher dosage of tgAAC009. Ninety-one healthy HIV-seronegative adults from three African countries were given one of three dosage levels of tgAAC09 (3 x 10(10), 3 x 10(11), or 3 x 10(12) DRP) intramuscularly, either at a 6- or 12-month interval; follow-up was 18 months. Overall, 65% and 57% of vaccine recipients experienced local and systemic signs and symptoms, respectively, most being mild. Frequency and severity were not dose related and were similar to those in placebo recipients. No vaccine-related serious adverse events were reported. Overall, HIV-specific T cell responses were detected by IFN-gamma ELISPOT in 17/69 (25%) vaccine recipients with 38% (10/26) responders in the highest dosage group. The response rate improved significantly with boosting at 6, but not 12 months, in the 3 x 10(11) and 3 x 10(12) dosage groups only. Neutralizing antibody titers to the AAV2 did not alter the frequency of immune responses to HIV. Two doses of tgAAC09 were well tolerated at the dosage levels given. Fewer than half the recipients of the highest vaccine dosage, 3 x 10(12) DRP, had T cell responses to HIV.

Gene therapy for late infantile neuronal ceroid lipofuscinosis: neurosurgical considerations

The authors conducted a phase I study of late infantile neuronal ceroid lipofuscinosis using an adenoassociated virus serotype 2 (AAV2) vector containing the deficient CLN2 gene (AAV2(CU)hCLN2). The operative technique, radiographic changes, and surgical complications are presented. Ten patients with late infantile neuronal ceroid lipofuscinosis disease each underwent infusion of AAV2(CU)hCLN2 (3 x 10(12) particle units) into 12 distinct cerebral locations (2 depths/bur hole, 75 minutes/infusion, and 2 microl/minute). Innovative surgical techniques were developed to overcome several obstacles for which little or no established techniques were available. Successful infusion relied on preoperative stereotactic planning to optimize a parenchymal target and diffuse administration. Six entry sites, each having 2 depths of injections, were used to reduce operative time and enhance distribution. A low-profile rigid fixation system with 6 integrated holding arms was utilized to perform simultaneous infusions within a practical time frame. Dural sealant with generous irrigation was used to avoid CSF egress with possible subdural hemorrhage or altered stereotactic registration. Radiographically demonstrated changes were seen in 39 (65%) of 60 injection sites, confirming localization and infusion. There were no radiographically or clinically defined complications. The neurosurgical considerations and results of this study are presented to offer guidance and a basis for the design of future gene therapy or other clinical trials in children that utilize direct therapeutic delivery.

The carboxy‐terminal fragment of inhibitor‐2 of protein phosphatase‐2A induces Alzheimer disease pathology and cognitive impairment

Development of rational therapeutic treatments of Alzheimer disease (AD) requires the elucidation of the etiopathogenic mechanisms of neurofibrillary degeneration and β-amyloidosis, the two hallmarks of this disease. Here we show, employing an adeno-associated virus serotype 1 (AAV1)-induced expression of the C-terminal fragment (I(2CTF)) of I(2)(PP2A), also called SET, in rat brain, decrease in protein phosphatase 2A (PP2A) activity, abnormal hyperphosphorylation of tau, and neurodegeneration; littermates treated identically but with vector only, i.e., AAV1-enhanced green fluorescent protein (GFP), served as a control. Furthermore, there was an increase in the level of activated glycogen synthase kinase-3β and enhanced expression of intraneuronal Aβ in AAV1-I(2CTF) animals. Morris water maze behavioral test revealed that infection with AAV1-I(2CTF) induced spatial reference memory and memory consolidation deficits and a decrease in the brain level of pSer133-CREB. These findings suggest a novel etiopathogenic mechanism of AD, which is initiated by the cleavage of I(2)(PP2A), producing I(2CTF), and describe a novel disease-relevant nontransgenic animal model of AD.

A novel etiopathogenic mechanism and a disease-relevant non-transgenic rat model of Alzheimer's disease

Alzheimer's disease (AD) is pathologically characterized by the presence of numerous intracellular neurofibrillary tangles and extracellular β-amyloid plaques. Development of rational therapeutical treatments of AD requires the elucidation of the mechanisms that lead to tau protein pathology and β-amyloidosis. We employed an adeno-associated virus serotype 1 (AAV1)-induced expression of the C-terminal fragment (I2CTF) of I2PP2A/SET and infected newborn Wistar rat pups by bilateral intracerebroventricular (ICV) injection with the AAV1- I2CTF. I2CTF, tau phosphorylation and neurodegeneration were studied immunohistochemically and by Western blot. The reference memory of the animals was evaluated by a spatial reference memory task in the water maze. The AAV1- I2CTF induced a decrease in brain protein phosphatase 2A (PP2A) activity, abnormal hyperphosphorylation of tau and neurodegeneration. Furthermore, there was an increase in the level of activated glycogen synthase kinase-3β and enhanced expression of intraneuronal Aβ in AAV1-I2CTF animals. Morris Water Maze behavioral test revealed that infection with AAV1-I2CTF induced a spatial reference memory and memory consolidation deficit. Finally, this cognitive impairment was associated with a decrease in the brain level of pSer133-CREB. This findings of this study suggest (1) a novel etiopathogenic mechanism of AD which is initiated by the cleavage of I2PP2A, producing I2CTF and (2) provide a practical, non-transgenic animal model which is based on an etiopathogenic mechanism of AD.

AAV Provides an Alternative for Gene Therapy of the Peripheral Sensory Nervous System

Recombinant adeno-associated virus (rAAV) is a promising vector for applications in the central nervous system (CNS). Clinical phase I/II studies have demonstrated safety of rAAV-expressing therapeutic transgenes for a variety of benign (i.e., noncancer) CNS disorders, namely, Canavan disease,1 Parkinson’s disease,2,3,4 and Batten disease.5 Human studies were also recently reported targeting the retina.6,8,9,9 From the early stages of preclinical development, rAAV was appealing because it was found to be neurotrophic and capable of long-term activity without immune interference or toxicity when injected into the CNS.10