Of the adeno-associated viruses (AAVs), AAV9 is known for its capability to cross the blood-brain barrier (BBB) and can, therefore, be used as a noninvasive method to target the central nervous system. Furthermore, the addition of the peptide PhP.B to AAV9 increases its transduction across the BBB by 40-fold. Another neurotropic serotype, AAV5, has been shown as a gene therapeutic delivery vehicle to ameliorate several neurodegenerative diseases in preclinical models, but its administration requires invasive surgery. In this study, AAV9-PhP.B and AAV5-PhP.B were designed and produced in an insect cell-based system. To AAV9, the PhP.B peptide TLAVPFK was added, whereas in AAV5-PhP.B (AQTLAVPFKAQAQ), with AQ-AQAQ sequences used to swap with the corresponding sequence of AAV5. The addition of PhP.B to AAV5 did not affect its capacity to cross the mouse BBB, while increased transduction of liver tissue was observed. Then, intravenous (IV) and intrastriatal (IStr) delivery of AAV9-PhP.B and AAV5 were compared. For AAV9-PhP.B, similar transduction and expression levels were achieved in the striatum and cortex, irrespective of the delivery method used. IStr administration of AAV5 resulted in significantly higher amounts of vector DNA and therapeutic miRNA in the target regions such as striatum and cortex when compared with an IV administration of AAV9-PhP.B. These results illustrate the challenge in developing a vector that can be delivered noninvasively while achieving a transduction level similar to that of direct administration of AAV5. Thus, for therapeutic miRNA delivery with high local expression requirements, intraparenchymal delivery of AAV5 is preferred, whereas a humanized AAV9-PhP.B may be useful when widespread brain (and peripheral) transduction is needed.

Striatal delivery of microRNA-gene therapy results in widespread brain huntingtin protein lowering in Huntington’s disease minipigs up to 1 year.

Gene therapy is being developed for the treatment of inherited diseases, whereby a therapeutic gene is continuously expressed in patients after delivery via viral vectors such as adeno-associated virus (AAV). Depending on the transgene, there could be a limited therapeutic window, and regulating timing and levels of transgene expression is advantageous. To control transgene transcription, the regulatory system GeneSwitch (GS) was evaluated in detail both in vitro and in vivo. The classical two-plasmid mifepristone (MFP)-inducible GS system was put into one plasmid or a single AAV5 vector. Our data demonstrate the inducibility of multiple transgenes and the importance of promoter and regulatory elements within the GS system. Mice injected with AAV5 containing the GS system transiently expressed mRNA and protein after MFP induction. The inducer MFP could be measured in plasma and liver tissue, and assessment of MFP and its metabolites showed rapid clearance from murine plasma. In a head-to-head comparison, our single vector outclassed the classical two-vector GS system. Finally, we show repeated inducibility of the transgene that also translated into a dynamic phenotypic change in mice. Taken together, this in-depth analysis of the GS system shows its applicability for regulated gene therapy.

Contemporary cochlear implants (CI) are generally very effective for remediation of severe to profound sensorineural hearing loss, but outcomes are still highly variable. Auditory nerve survival is likely one of the major factors underlying this variability. Neurotrophin therapy therefore has been proposed for CI recipients, with the goal of improving outcomes by promoting improved survival of cochlear spiral ganglion neurons (SGN) and/or residual hair cells. Previous studies have shown that glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor, and neurotrophin-3 can rescue SGNs following insult. The current study was designed to determine whether adeno-associated virus vector serotype 5 (AAV-5) encoding either green fluorescent protein or GDNF can transduce cells in the mouse cochlea to express useful levels of neurotrophin and to approximate the optimum therapeutic dose(s) for transducing hair cells and SGN. The findings demonstrate that AAV-5 is a potentially useful gene therapy vector for the cochlea, resulting in extremely high levels of transgene expression in the cochlear inner hair cells and SGN. However, overexpression of human GDNF in newborn mice caused severe neurological symptoms and hearing loss, likely due to Purkinje cell loss and cochlear nucleus pathology. Thus, extremely high levels of transgene protein expression should be avoided, particularly for proteins that have neurological function in neonatal subjects.

May 6, 2019April 9, 2019Free AccessThe Biodistribution and Tolerability of rAAV5-miHTT after Bilateral Intra-striatal Delivery to Non-human Primates (S16.006)Martin de Haan, Bas Blits, Lisa Spronck, Astrid Valles-Sanchez, Melvin Evers, Sander van Deventer, Pavlina Kostantinova, and Joseph HigginsAuthors Info & AffiliationsApril 9, 2019 issue92 (15_supplement)https://doi.org/10.1212/WNL.92.15_supplement.S16.006 Letters to the Editor

Perineuronal nets (PNNs) are condensed structures in the extracellular matrix that mainly surround GABA-ergic parvalbumin-positive interneurons in the adult brain. Previous studies revealed a parallel between PNN formation and the closure of the critical period. Moreover, ocular dominance plasticity is enhanced in response to PNN manipulations in adult animals. However, the mechanisms through which perineuronal nets modulate plasticity are still poorly understood. Recent work indicated that perineuronal nets may convey molecular signals by binding and storing proteins with important roles in cellular communication. Here we report that semaphorin3A (Sema3A), a chemorepulsive axon guidance cue known to bind to important perineuronal net components, is necessary to dampen ocular dominance plasticity in adult rats. First, we showed that the accumulation of Sema3A in PNNs in the visual cortex correlates with critical period closure, following the same time course of perineuronal nets maturation. Second, the accumulation of Sema3A in perineuronal nets was significantly reduced by rearing animals in the dark in the absence of any visual experience. Finally, we developed and characterized a tool to interfere with Sema3A signaling by means of AAV-mediated expression of receptor bodies, soluble proteins formed by the extracellular domain of the endogenous Sema3A receptor (neuropilin1) fused to a human IgG Fc fragment. By using this tool to antagonize Sema3A signaling in the adult rat visual cortex, we found that the specific inhibition of Sema3A promoted ocular dominance plasticity. Thus, Sema3A accumulates in perineuronal nets in an experience-dependent manner and its presence in the mature visual cortex inhibits plasticity.

BackgroundHTT-lowering therapies hold great promise to slow-down or halt neurodegeneration in Huntington disease (HD). We have developed an engineered microRNA targeting human huntingtin (HTT), delivered via adeno-associated viral vector serotype 5 (AAV5-miHTT), leading to efficient HTT-lowering in vitro and in vivo in rodent models.AimTo assess the translatability of our approach in a large animal model: transgenic HD (tgHD) minipigs.MethodsAnimals were injected with AAV5-miHTT (1.2 × 1013 gc/brain), bilaterally into striatum (caudate and putamen) and sacrificed 6 months post-treatment. Across different brain regions, vector DNA, miHTT and mutant HTT (mHTT) mRNA were measured by Q-PCR, and mHTT protein using an ultrasensitive immunoassay. In longitudinal cerebrospinal fluid (CSF) samples, miHTT and mHTT protein expression were assessed by Q-PCR and ultrasensitive immunoassay, respectively.ResultsWidespread brain biodistribution of vector DNA was observed, with the highest levels in target (striatal) regions but also in thalamus and cortical regions, in both grey and white matter. Expression of miHTT was highly correlated with vector DNA in all brain areas. Corresponding to the vector DNA and miHTT expression, a reduction of mHTT mRNA and protein was observed in AAV5-miHTT treated animals with respect to controls. mHTT protein lowering was on average more than 75% in the injected areas, and between 30–50% in most of the distal regions. Translational pharmacokinetic and pharmacodynamic measures in the CSF were in line with the effects observed in the brain. We detected CSF miHTT, and CSF mHTT protein lowering up to 50% at 3 and 70% at 6 months post-dosing.ConclusionsThis study demonstrates widespread biodistribution and durable efficiency of AAV5-miHTT in disease-relevant regions in a large brain, and the potential of CSF translational measures to follow-up efficacy.

Background Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Great effort has been put in proof-of-concept studies of therapeutic agents in HD rodent models. One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particular relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Aims Here, we investigated the feasibility, efficacy, and tolerability of huntingtin-lowering gene therapy in a large animal brain. Methods Transgenic HD (tgHD) minipigs were injected with an engineered microRNA targeting human huntingtin, delivered via adeno-associated viral vector serotype 5 (AAV5-miHTT) or AAV5-GFP as control. The viruses were intracranially administered into the striatum and thalamus. Results We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. Conclusion The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.

Many therapeutic strategies aimed at relieving symptoms of Parkinsons disease (PD) are currently used for treatment of this disease. With a hallmark of progressive degeneration of dopaminergic neurons, the absence of properly operational dopaminergic circuitry becomes a therapeutic target. Following diagnosis, dopamine replacement can be given in the form of L-DOPA (L-3,4-dihydroxyphenylalanine). Even though it is recognized as standard of care, this treatment strategy does not prevent the affected neurons from degenerating. Therefore, studies have been performed using gene therapy (GT) to make dopamine (DA) available from within the brain using an artificial DA circuitry. One approach is to administer a GT aimed at delivering the key enzymes for DA synthesis using a lentiviral vector system (Palfi et al., 2014). A similar approach has been investigated with adeno-associated virus (AAV) expressing aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP-cyclohydrolase I (Bankiewicz et al., 2000), which are downregulated in PD. Another GT approach to mitigate symptoms of PD used AAV-mediated delivery of GAD-67 (glutamate decarboxylase) (Kaplitt et al., 2007). This approach mimics the inhibitory effect of DA neurons on their targets, in reducing motor abnormalities. Finally, disease modifying strategies have been undertaken using neurotrophic factors such as neurturin (NTN) (Marks et al., 2008; Bartus et al., 2013a) or are ongoing with the closely related Glial cell line-derived neurotrophic factor. Those approaches are aiming at rescuing the degenerating neurons. All of the above mentioned strategies have their own merits, but also some disadvantages. So far, none of clinical applied GT studies has resulted in significant clinical benefit, although some clinical studies are ongoing and results are expected over the next few years.

not available at time of printing.

This study was performed to investigate the delivery of AAV vectors into the putamen and thalamus of nonhuman primates. The vectors were delivered to the targeted brain regions by MRI-guided convection enhanced delivery. Special attention was given to the analysis of axonal transport and levels of transduction. Recombinant AAV is an excellent candidate for delivery of therapeutic molecules to the central nervous system to target neurodegenerative diseases. UniQure has succeeded in developing a proprietary platform manufacturing technology that allows safe, effective, cGMP-compliant, economically feasible and commercially scalable manufacturing of AAV. UniQure's novel approach is based on the use of a combination of recombinant baculoviruses and insect cells. Using our production platform, two AAV stocks encoding GDNF or GFP were generated. At eight weeks following infusion into the thalamus, for instance, massive transduction of the thalamus, cortex, striatum and substantia nigra was observed indicating both anterograde and retrograde transduction. At the site of injection, transduction was both glial and neuronal, whereas off site transduction was mainly neuronal. Following injection of a lower AAV volume into the putamen, transduction was limited to areas within the putamen and substantia nigra suggesting only anterograde transport of viral particles. These data indicate a dose dependent anterograde or retrograde transport mechanism. This data set confirms that production of AAV using the (scalable) baculovirus-based platform results in an effective vector that is able to mediate expression patterns that can be used to develop an AAV-mediated therapeutic strategy to treat neurodegenerative diseases.

The most upstream therapeutic target in Huntington’s disease (HD) is the mutated huntingtin (mtHTT) and gene silencing with artificial miRNAs (miHTTs) delivered by adeno-associated viral vector (AAV) is expected to have therapeutic benefit. We have taken two approaches for the development of RNAi-based gene therapy of HD: total HTT silencing by targeting exon 1 and allele-specific inhibition by targeting heterozygous SNPs or a deletion linked to mtHTT. Anti-HTT target sequences were incorporated in different pri-miRNA scaffolds and their knockdown efficacy, allele selectivity and pri-miHTT processing were analysed in vitro. The best miHTT candidates were incorporated in AAV5 vector and produced using the established uniQure baculovirus-based manufacturing platform. Proof of concept studies have shown efficacy of AAV5-miHTT in the lentivirally-derived HD rat model and in the humanised HD mouse model. In both models, AAV5-miHTT delivery resulted in a reduction of the disease-related HTT protein which was associated with a delay of neurodegeneration and in reduction of mtHTT aggregates. Furthermore, the miHTT processing, safety and off-target potential are being determined by next generation sequencing (NGS) on RNA isolated from murine striata and cortices to support the selection of the therapeutic candidate for clinical development. These preclinical results suggest that AAV5-miHTT may provide important therapeutic benefit for the HD patients and will allow for long-term HTT suppression upon a single vector administration.

Widespread distribution of gene products at clinically relevant levels throughout the central nervous system (CNS) without involving peripheral organs is still a challenge. Previous work demonstrated that infusion of adeno-associated virus (AAV) serotypes 9 (AAV9) and 7 (AAV7) into the cerebrospinal fluid (CSF) via cisterna magna of nonhuman primates (NHP) resulted in widespread transduction throughout cortex and spinal cord. Adeno-associated virus serotype 5 (AAV5) is also a potential candidate for CNS gene therapy when delivered into brain parenchyma but its performance in CSF has not been described. In the present work, we compared transduction patterns after CSF and parenchymal delivery of AAV5-GFP. For CSF delivery, we injected various doses of AAV5 (1E+14, 1E+13 and 1E+12 vg/mL) into CSF via lumbar routes in naïve NHP and studied vector distribution and cellular transduction 4 weeks after CSF delivery. We found a dose-dependent increase in transduction with strong levels of cell transduction and distribution throughout cortex and along the spinal cord at the highest doses and no signal in the low-dose group. These results suggest a dose threshold when delivering into CSF, most likely due to a dilution of the viral particles. Both astrocytes and neurons were transduced by AAV5 when infused either in CSF or directly into brain parenchyma. Parenchymal delivery of AAV5-GFP was analyzed 8 weeks after surgery. Interestingly, when infused into the thalamus and the contralateral putamen, AAV5 resulted in higher levels of expression in cortical and subcortical structures than those observed in high-dose CSF animals. Surprisingly, thalamic injection directed spinal cord transduction throughout the cortico-bulbar axis. These results suggest not only that smaller volumes of AAV5 infused directly into the thalamus can result in robust and widespread cellular transduction in the brain but also to spinal cord. Our results confirm AAV5 as a powerful viral vector for gene transfer in the CNS and underscore its translational potential for treating neurological disorders of the brain and spinal cord.

Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan’s, Parkinson’s (PD) and Alzheimer’s disease (AD), retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral vectors (AAV) and the first AAV-based therapeutic, a vector encoding lipoprotein lipase, is now marketed in Europe under the name Glybera. These remarkable advances may become relevant to translational research on gene therapy to promote peripheral nervous system (PNS) repair. This short review first summarizes the results of gene therapy in animal models for peripheral nerve repair. Secondly, we identify key areas of future research in the domain of PNS-gene therapy. Finally, a perspective is provided on the path to clinical translation of PNS-gene therapy for traumatic nerve injuries. In the latter section we discuss the route and mode of delivery of the vector to human patients, the efficacy and safety of the vector, and the choice of the patient population for a first possible proof-of-concept clinical study.

Parkinson’s disease (PD) is characterized by neurodegeneration of the dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) has been identified as possible therapeutic molecule for the treatment of neurodegenerative diseases in several different animal models. Delivery of the GDNF has been proven to be very efficient using recombinant AAV vectors. AAV2 has been widely used for the delivery of transgenes to the brain and has even led to a clinical trial for the treatment of PD. A serotype that is known for highly effective delivery of its transgene to the brain is AAV serotype 5. At uniQure, we have developed a baculoviral-based triple infection method of SF9 insect cells that is scalable for GMP use. Using this method two AAV5 stocks encoding GDNF or GFP under control of the CAG promoter were generated and used in the current study. Biological activity of the vector was demonstrated in vitro using conditioned medium from transduced cells. In vivo analysis of the vector was performed in healthy rats following slow infusion into the brains. Recombinant AAV-mediated GDNF was detected in brain homogenates and on tissue sections. When the vector was infused in a 6-OHDA rat model, the GDNF was able to almost completely rescue the neurons in the substantia nigra. These results indicate that our viral vector can be used in an in vivo setting and can be tested in neurodegenerative disease models and further developed for possible clinical testing.

BACKGROUND:The efficacy and safety of intracerebral gene therapy for brain disorders like Parkinson's disease depends on the appropriate distribution of gene expression.OBJECTIVES:To assess whether the distribution of gene expression is affected by vector titer and protein type.METHODS:Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received a 30-µl inoculation of a high- or a low-titer suspension of AAV5 encoding glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP) in the right and left ventral postcommissural putamen. The inoculations were conducted using convection-enhanced delivery and intraoperative MRI (IMRI).RESULTS:IMRI confirmed targeting and infusion cloud irradiation from the catheter tip into the surrounding area. A postmortem analysis 6 weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection site that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the substantia nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many neurons of the SNpc and SNpr.CONCLUSIONS:After controlling for target and infusate volume, the intracerebral distribution of the gene product was affected by the vector titer and product biology.