Enhanced Cochlear Transduction by AAV9 with High-Concentration Sucrose.

The inner ear is a primary lesion in sensorineural hearing loss and has been a target in gene therapy. The efficacy of gene therapy depends on achieving sufficient levels of transduction at a safe vector dose. Vectors derived from various adeno-associated viruses (AAVs) are predominantly used to deliver therapeutic genes to inner ear cells. AAV9 and its variants vector are attractive candidates for clinical applications since they can cross the mesothelial cell layer and transduce inner hair cells (IHCs), although this requires relatively high doses. In this study, we investigated the effects of sucrose on the transduction of a variant of the AAV9 vector for gene transfer in the inner ear. We found that high concentrations of sucrose increased gene transduction in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells in vitro. In addition, we demonstrated that simultaneous administration of sucrose enhanced the transduction of mouse IHCs and spiral ligament cells using an AAV9 variant vector. The procedure did not increase the thresholds in the auditory brainstem response, suggesting that sucrose had no adverse effect on auditory function. This versatile method may be valuable in the development of novel gene therapies for adult-onset sensorineural hearing loss.

Early distribution of18 F-labeled AAV9 vectors in the cerebrospinal fluid after intracerebroventricular or intracisternal magna infusion in non-human primates.

BACKGROUND: The delivery of adeno-associated virus (AAV) vectors via the cerebrospinal fluid (CSF) has emerged as a valuable method for widespread transduction in the central nervous system. Although infusion into the cerebral ventricles is a common protocol in preclinical studies of small animals, the cisterna magna has been recognized as an alternative target for clinical studies because it can be reached in a less invasive manner using an intrathecal catheter via the subarachnoid space from a lumbar puncture. METHODS: We evaluated the early distribution of fluorine-18-labeled AAV9 vectors infused into the lateral ventricle or cisterna magna of four non-human primates using positron emission tomography. The expression of the green fluorescent protein was immunohistochemically determined. RESULTS: In both approaches, the labeled vectors diffused into the broad arachnoid space around the brain stem and cervical spinal cord within 30 min. Both infusion routes efficiently transduced neurons in the cervical spinal cord. CONCLUSIONS: For gene therapy that primarily targets the cervical spinal cord and brainstem, such as amyotrophic lateral sclerosis, cisterna magna infusion would be a feasible and effective administration method.

Engineered adeno-associated virus 3 vector with reduced reactivity to serum antibodies.

The natural serotypes of adeno-associated virus (AAV) or their variants, such as AAV8 and AAV5, are commonly used as vectors in the clinical programs for liver-targeted gene therapy. While AAV8 vectors are not highly efficient at targeting primary human hepatocytes, AAV3 vectors have recently demonstrated remarkable efficiency at targeting both human and non-human primate hepatocytes. However, the presence of high levels of neutralizing antibodies (NAbs) impedes transduction into hepatocytes, representing a major obstacle to the clinical application of AAV3 vectors. Herein, we engineered the viral capsid to reduce its reactivity with pre-existing NAbs, thereby enhancing the transduction efficiency. By introducing three substitutions (S472A, S587A, and N706A) on the surface loop of AAV3B capsid protein, we generated a triple mutant AAV3 (AAV.GT5) vector with less reactivity to anti-AAV capsid NAbs. While the transduction efficiency of AAV.GT5 into human hepatocellular cell lines was similar to those of parental AAV3B, it was 50-fold higher for hepatocytes derived from humanized mice compared to AAV8 vectors. Moreover, the AAV.GT5 vector yield was similar to those of the AAV2 and AAV3B vectors. Thus, high resistance to pre-existing NAbs makes AAV.GT5 a promising candidate for future liver-targeted gene therapy clinical trials.

Intra-cisterna magna delivery of an AAV vector with the GLUT1 promoter in a pig recapitulates the physiological expression of SLC2A1.

Glucose transporter 1 deficiency syndrome (GLUT1DS) is caused by haplo-insufficiency of SLC2A1, which encodes GLUT1, resulting in impaired hexose transport into the brain. Previously, we generated a tyrosine-mutant AAV9/3 vector in which SLC2A1 was expressed under the control of the endogenous GLUT1 promoter (AAV-GLUT1), and confirmed the improved motor function and cerebrospinal fluid glucose levels of Glut1-deficient mice after cerebroventricular injection of AAV-GLUT1. In preparation for clinical application, we examined the expression of transgenes after intra-cisterna magna injection of AAV-GFP (tyrosine-mutant AAV9/3-GFP with the CMV promoter) and AAV-GLUT1. We injected AAV-GFP or AAV-GLUT1 (1.63 × 1012 vector genomes/kg) into the cisterna magna of pigs to compare differential promoter activity. After AAV-GFP injection, exogenous GFP was expressed in broad areas of the brain and peripheral organs. After AAV-GLUT1 injection, exogenous GLUT1 was expressed predominantly in the brain. At the cellular level, exogenous GLUT1 was mainly expressed in the endothelium, followed by glia and neurons, which was contrasted with the neuronal-predominant expression of GFP by the CMV promotor. We consider intra-cisterna magna injection of AAV-GLUT1 to be a feasible approach for gene therapy of GLUT1DS.

Alzheimer Aß Assemblies Accumulate in Excitatory Neurons upon Proteasome Inhibition and Kill Nearby NAKα3 Neurons by Secretion.

We identified ∼30-mer amyloid-ß protein (Aß) assemblies, termed amylospheroids, from brains of patients with Alzheimer disease (AD) as toxic entities responsible for neurodegeneration and showed that Na+,K+-ATPase α3 (NAKα3) is the sole target of amylospheroid-mediated neurodegeneration. However, it remains unclear where in neurons amylospheroids form and how they reach their targets to induce neurodegeneration. Here, we present an in vitro culture system designed to chronologically follow amylospheroid formation in mature neurons expressing amyloid precursor protein bearing early-onset AD mutations. Amylospheroids were found to accumulate mainly in the trans-Golgi network of excitatory neurons and were initially transported in axons. Proteasome inhibition dramatically increased amylospheroid amounts in trans-Golgi by increasing Aß levels and induced dendritic transport. Amylospheroids were secreted and caused the degeneration of adjacent NAKα3-expressing neurons. Interestingly, the ASPD-producing neurons later died non-apoptotically. Our findings demonstrate a link between ASPD levels and proteasome function, which may have important implications for AD pathophysiology.

Gene therapy for Glut1-deficient mouse using an adeno-associated virus vector with the human intrinsic GLUT1 promoter.

BACKGROUND: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene, encoding glucose transporter type 1 (GLUT1), was expressed under the human endogenous GLUT1 promoter (AAV-GLUT1). We examined whether AAV-GLUT1 administration could lead to functional improvement in GLUT1-deficient mice. METHODS: We extrapolated human endogenous GLUT1 promoter sequences from rat minimal Glut1 promoter sequences. We generated a tyrosine-mutant AAV9/3 vector in which human SLC2A1-myc-DDK was expressed under the human GLUT1 promoter (AAV-GLUT1). AAV-GLUT1 was administered to GLUT1-deficient mice (GLUT1+/- mice) via intracerebroventricular injection (1.85 × 1010 vg/mouse or 6.5 × 1010 vg/mouse). We analyzed exogenous GLUT1 mRNA and protein expression in the brain and other major organs. We also examined improvements of cerebral microvasculature, motor function using rota-rod and footprint tests, as well as blood and cerebrospinal fluid (CSF) glucose levels. Additionally, we confirmed exogenous GLUT1 protein distribution in the brain and other organs after intracardiac injection (7.8 × 1011 vg/mouse). RESULTS: Exogenous GLUT1 protein was strongly expressed in the cerebral cortex, hippocampus and thalamus. It was mainly expressed in endothelial cells, and partially expressed in neural cells and oligodendrocytes. Motor function and CSF glucose levels were significantly improved following intracerebroventricular injection. Exogenous GLUT1 expression was not detected in other organs after intracerebroventricular injection of AAV-GLUT1, whereas it was detected in the liver and muscle tissue after intracardiac injection. CONCLUSIONS: Exogenous GLUT1 expression after AAV-GLUT1 injection approximated that of physiological human GLUT1 expression. Local central nervous system administration of AAV-GLUT1 improved CSF glucose levels and motor function of GLUT1-deficient mice and minimized off-target effects.

Persistent Expression of Dopamine-Synthesizing Enzymes 15 Years After Gene Transfer in a Primate Model of Parkinson's Disease.

Restoring dopamine production in the putamen through gene therapy is a straightforward strategy for ameliorating motor symptoms for Parkinson's disease (PD). In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity-based primate model of PD, we previously showed the safety and efficacy of adeno-associated viral (AAV) vector-mediated gene delivery to the putamen of three dopamine-synthesizing enzymes (tyrosine hydroxylase [TH], aromatic l-amino acid decarboxylase [AADC], and guanosine triphosphate cyclohydrolase I [GCH]) up to 10 months postprocedure. Although three of four monkeys in this study have previously undergone postmortem analysis, one monkey was kept alive for 15 years after gene therapy to evaluate long-term effects. Here, we report that this monkey showed behavioral recovery in the right-side limb that remained unchanged for 15 years, at which time euthanasia was carried out owing to onset of senility. Immunohistochemistry of the postmortem brain from this monkey revealed persistent expression of TH, AADC, and GCH genes in the lesioned putamen. Transduced neurons were broadly distributed, with the estimated transduction region occupying 91% of the left postcommissural putamen. No signs of cytotoxicity or Lewy body pathology were observed in the AAV vector-injected putamen. This study provides evidence of long-term safety and efficacy of the triple-transduction method as a gene therapy for PD.

Gene therapy for a mouse model of glucose transporter-1 deficiency syndrome.

OBJECTIVE: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene was expressed under the synapsin I promoter (AAV-hSLC2A1) and examined if AAV-hSLC2A1 administration can lead to functional improvement in GLUT1-deficient mice. METHODS: AAV-hSLC2A1 was injected into heterozygous knock-out murine Glut1 (GLUT1+/-) mice intraperitoneally (systemic; 1.85 × 1011 vg/mouse) or intra-cerebroventricularly (local; 1.85 × 1010 vg/mouse). We analyzed GLUT1 mRNA and protein expression, motor function using rota-rod and footprint tests, and blood and cerebrospinal fluid (CSF) glucose levels. RESULTS: Vector-derived RNA was detected in the cerebrum for both injection routes. In the intra-cerebroventricular injection group, exogenous GLUT1 protein was strongly expressed in the cerebral cortex and hippocampus near the injection site. In the intraperitoneal injection group, exogenous GLUT1 protein was mildly expressed in neural cells throughout the entire central nervous system. The motor function test and CSF/blood glucose ratio were significantly improved following intra-cerebroventricular injection. CONCLUSIONS: AAV-hSLC2A1 administration produced exogenous GLUT1 in neural cells and improved CSF glucose levels and motor function of heterozygous knock-out murine Glut1 mice.

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-ß assembly.

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aß-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.

Systemic delivery of tyrosine-mutant AAV vectors results in robust transduction of neurons in adult mice.

Recombinant adeno-associated virus (AAV) vectors are powerful tools for both basic neuroscience experiments and clinical gene therapies for neurological diseases. Intravascularly administered self-complementary AAV9 vectors can cross the blood-brain barrier. However, AAV9 vectors are of limited usefulness because they mainly transduce astrocytes in adult animal brains and have restrictions on foreign DNA package sizes. In this study, we show that intracardiac injections of tyrosine-mutant pseudotype AAV9/3 vectors resulted in extensive and widespread transgene expression in the brains and spinal cords of adult mice. Furthermore, the usage of neuron-specific promoters achieved selective transduction of neurons. These results suggest that tyrosine-mutant AAV9/3 vectors may be effective vehicles for delivery of therapeutic genes, including miRNAs, into the brain and for treating diseases that affect broad areas of the central nervous system.

Multitracer assessment of dopamine function after transplantation of embryonic stem cell-derived neural stem cells in a primate model of Parkinson's disease.

The ability of primate embryonic stem (ES) cells to differentiate into dopamine (DA)-synthesizing neurons has raised hopes of creating novel cell therapies for Parkinson's disease (PD). As the primary purpose of cell transplantation in PD is restoration of dopaminergic neurotransmission in the striatum, in vivo assessment of DA function after grafting is necessary to achieve better therapeutic effects. A chronic model of PD was produced in two cynomolgus monkeys (M-1 and M-2) by systemic administration of neurotoxin. Neural stem cells (NSCs) derived from cynomolgus ES cells were implanted unilaterally in the putamen. To evaluate DA-specific functions, we used multiple [(11)C]-labeled positron emission tomography (PET) tracers, including [beta-(11)C]L-3,4-dihydroxyphenylalanine (L-[beta-(11)C]DOPA, DA precursor ligand), [(11)C]-2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(11)C]beta-CFT, DA transporter ligand) and [(11)C]raclopride (D(2) receptor ligand). At 12 weeks after grafting NSCs, PET demonstrated significantly increased uptake of L-[beta-(11)C]DOPA (M-1:41%, M-2:61%) and [(11)C]beta-CFT (M-1:31%, M-2:36%) uptake in the grafted putamen. In addition, methamphetamine challenge in M-2 induced reduced [(11)C]raclopride binding (16%) in the transplanted putamen, suggesting release of DA. These results show that transplantation of NSCs derived from cynomolgus monkey ES cells can restore DA function in the putamen of a primate model of PD. PET with multitracers is useful for functional studies in developing cell-based therapies against PD.

Viral-mediated temporally controlled dopamine production in a rat model of Parkinson disease.

Regulation of gene expression is necessary to avoid possible adverse effects of gene therapy due to excess synthesis of transgene products. To reduce transgene expression, we developed a viral vector-mediated somatic regulation system using inducible Cre recombinase. A recombinant adeno-associated virus (AAV) vector expressing Cre recombinase fused to a mutated ligand-binding domain of the estrogen receptor alpha (CreER(T2)) was delivered along with AAV vectors expressing dopamine-synthesizing enzymes to rats of a Parkinson disease model. Treatment with 4-hydroxytamoxifen, a synthetic estrogen receptor modulator, activated Cre recombinase within the transduced neurons and induced selective excision of the tyrosine hydroxylase (TH) coding sequence flanked by loxP sites, leading to a reduction in transgene-mediated dopamine synthesis. Using this strategy, aromatic L-amino acid decarboxylase (AADC) activity was retained so that l-3,4-dihydroxyphenylalanine (L-dopa), a substrate for AADC, could be converted to dopamine in the striatum and the therapeutic effects of L-dopa preserved, even after reduction of TH expression in the case of dopamine overproduction. Our data demonstrate that viral vector-mediated inducible Cre recombinase can serve as an in vivo molecular switch, allowing spatial and temporal control of transgene expression, thereby potentially increasing the safety of gene therapy.