BackgroundAs a step towards clinical use of AAV-mediated gene therapy, brains of large animals are used to settle delivery parameters as most brain connections, and relative sizes in large animals and primates, are reasonably common. Prior to application in the clinic, approaches that have shown to be successful in rodent models are tested in larger animal species, such as dogs, non-human primates, and in this case, minipigs.New methodWe evaluated alternate delivery routes to target the basal ganglia by injections into the more superficial corona radiata, and, deeper into the brain, the thalamus. Anatomically known connections can be used to predict the expression of the transgene following infusion of AAV5. For optimal control over delivery of the vector with regards to anatomical location in the brain and spread in the tissue, we have used magnetic resonance image-guided convection-enhanced diffusion delivery.ResultsWhile the transduction of the cortex was observed, only partial transduction of the basal ganglia was achieved via the corona radiata. Thalamic administration, on the other hand, resulted in widespread transduction from the midbrain to the frontal cortexComparison with existing methodsCompared to other methods, such as delivery directly to the striatum, thalamic injection may provide an alternative when for instance, injection into the basal ganglia directly is not feasible.ConclusionsThe study results suggest that thalamic administration of AAV5 has significant potential for indications where the transduction of specific areas of the brain is required.

Various administration routes of adeno-associated virus (AAV)-based gene therapy have been examined to target the central nervous system to answer the question what the most optimal delivery route is for treatment of the brain with certain indications. In this study, we evaluated AAV5 vector system for its capability to target the central nervous system via intrastriatal, intrathalamic or intracerebroventricular delivery routes in rats. AAV5 is an ideal candidate for gene therapy because of its relatively low level of existing neutralizing antibodies compared to other serotypes, and its broad tissue and cell tropism. Intrastriatal administration of AAV5-GFP resulted in centralized localized vector distribution and expression in the frontal part of the brain. Intrathalamic injection showed transduction and gradient expression from the rostral brain into lumbar spinal cord, while intracerebroventricular administration led to a more evenly, albeit relatively superficially distributed, transduction and expression throughout the central nervous system. To visualize the differences between localized and intra-cerebral spinal fluid administration routes, we compared intrastriatal to intracerebroventricular and intrathecal administration of AAV5-GFP. Together, our results demonstrate that for efficient transgene expression, various administration routes can be applied.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). 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 particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD(tgHD) minipig model. 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. 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.

The present study was designed to characterize transduction of non-human primate brain and spinal cord with AAV5 viral vector after parenchymal delivery. AAV5-CAG-GFP (1 × 1013 vector genomes per milliliter (vg ml−1)) was bilaterally infused either into putamen, thalamus or with the combination left putamen and right thalamus. Robust expression of GFP was seen throughout infusion sites and also in other distal nuclei. Interestingly, thalamic infusion of AAV5 resulted in the transduction of the entire corticospinal axis, indicating transport of AAV5 over long distances. Regardless of site of injection, AAV5 transduced both neurons and astrocytes equally. Our data demonstrate that AAV5 is a very powerful vector for the central nervous system and has potential for treatment of a wide range of neurological pathologies with cortical, subcortical and/or spinal cord affection.

Introduction: Gene therapy offers long term solutions for chronic diseases, whereby the transgene is continuously expressed upon single vector administration. However in some cases it would be desirable to tightly regulate or switch off transgene expression. Methods: We are investigating regulated gene expression based on the mifepristone (MFP)-inducible GeneSwitch system. The GeneSwitch protein comprises yeast Gal4 DNA-binding domain, a human p65 activation domain and a MFP controlled domain derived from the human progesterone receptor. The classical GeneSwitch system consists of two expression cassettes on two separate vectors; one containing the GeneSwitch sequence and one containing the transgene. We compared this two-vector system to a single-vector system, where the two cassettes were put into one vector for efficacy in vitro and in vivo. Results: We show inducible expression of EPO, IGF and GNDF obtained in vitro upon addition of MFP to cells transfected with plasmids containing GeneSwitch and the gene expression cassette. The kinetics of EPO mRNA and protein expression followed a dose dependent fashion in the range of 0.1 to 10 nM MFP and reached a plateau at higher MFP concentrations. Surprisingly, the GeneSwitch protein expression decreased 48h after MFP induction. The indicibility of the single versus the two-vector system of GeneSwitch-EPO was compared. Both systems were equally inducible based on total amount of EPO produced in the presence of MFP and related to background expression in the absence of MFP. In vivo proof of concept was obtained for EPO in the liver. EPO is characterized by clear expression kinetics in plasma and raises blood hematocrit, hence provides a reliable in-life read-out for gene inducibility. Mice were injected with different doses of AAV5-AAT-GeneSwitch-EPO and gene expression was induced in two separate rounds at 4 and 8 weeks p.i. EPO plasma levels increased approximately 2-logs in the single or two-vector system-injected mice, compared to un-induced groups. Moreover in the absence of MFP background expression of EPO was lower in the single-vector system and hematocrit levels were unaffected. Measurements of MFP in tissue matrices and in plasma by mass spectrometry show the presence of MFP in plasma and liver, validating applicability of the GeneSwitch system in the liver. Conclusion: Overall, our data indicate that transgene expression can be repeatedly regulated in the liver using the GeneSwitch system and provides us with a novel AAV5 vector for further development.

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the first exon of the HTT gene. This CAG repeat expansion results in an expanded polyglutamine repeat in the huntingtin (HTT) protein, causing toxic gain-of-function and affecting numerous cellular processes. Lowering the expression of mutant HTT (gene silencing) and thereby reducing downstream toxic effects is hypothesised to be therapeutically beneficial. Gene therapy using viral vectors can induce long-term HTT silencing following single administration. The gene silencing therapy developed by uniQure is based on a microRNA targeting human HTT (miHTT). The DNA expression cassette encoding the miHTT is delivered to the cell using adeno-associated viral vector serotype 5 (AAV5-miHTT). After a thorough selection process, the therapeutic lead candidate AAV5-miHTT has been identified that induces strong and safe HTT silencing in vivo. Proof-of-concept (PoC) studies have shown that local injection of AAV5-miHTT in the striatum of a lentiviral HD rat model resulted in reduced number of HTT aggregates and subsequently prevention of striatal neuronal dysfunction. In the humanised (Hu)128/21 mouse model sustained target engagement was shown with up to 80% HTT silencing seven months post intra-striatal injection. No acute toxicity of AAV5-miHTT has been observed thus far, nor were deleterious effects of HTT silencing shown up to seven months post-treatment in Hu18/18 control mice. Currently, long-term therapeutic PoC studies to examine mitigation of HD phenotype after AAV5-miHTT administration are ongoing in various rodent HD models and transgenic HD mini pigs. To determine route of delivery in large brain, AAV5-GFP has been administered into the central nervous system of control mini pigs and non-human primates (NHP). Administration of AAV5-GFP into the putamen and thalamus showed widespread distribution of the vector in the caudate nucleus, putamen, globus pallidus and cortex. At present, efficacy, safety and tolerability of AAV5-miHTT are being evaluated in NHPs. The combination of widespread vector distribution, great target engagement, long-term expression and good safety profile allowed us to select AAV5-miHTT as lead candidate for HD gene therapy development.

Gene therapy is currently one of the most advanced approaches investigated for the treatment of Huntington’s disease (HD). There is a high unmet need for disease-modifying HD therapy since to date, no clinical trials have demonstrated treatment efficacy. The therapeutic target in HD is the mutated huntingtin (Htt) and the goal of the gene therapy is to silence the gene with miRNAs expressed from an AAV5 vector. Adeno-associated viral vector (AAV) expressing artificial miRNAs targeting the Htt gene is being developed by uniQure as an RNA interference (RNAi)-based gene therapy of HD. Three major approaches have been undertaken for the development of RNAi-based HD gene therapy: (1) total Htt knockdown by targeting exon 1, (2) targeting the CAG repeats in Htt exon 1 and (3) allele-specific inhibition of mutant Htt expression by targeting SNP rs362331 in exon 50 and SNP rs362307 in exon 67. Initially 100 artificial miRNAs targeting the wild-type and/or mutant Htt gene were designed and verified for their knock-down efficacy in vitro. In the first approach we identified the highly potent miHtt10 and miHtt12 targeting exon 1 therefore inducing strong total Htt knockdown. In the second approach, we tried to target the CAG repeats and discriminate between wt and mutant Htt based on differences in the secondary structure of the mRNA. In the third approach we identified miSNP50 and miSNP7 that specifically inhibited the mutant Htt allele while the wild-type Htt was mildly affected. Next, murine striata were transduced with AAV5-miHtt12, and AAV5-miSNP67 and a strong knockdown of Htt reporter gene was observed within 8 weeks post injection. Ongoing research aims to assess the neuroprotective effect of the non-selective and allele-specific miRNAs targeting Htt and expressed from AAV5 vectors in rodent HD models. Different viral delivery routes by direct intrastriatal injection or in the cerebrospinal fluid are being evaluated. Furthermore, the Htt RNAi silencing efficacy, safety and off-target potential are determined. AAV5 delivery of miRNAs targeting Htt provides a novel approach for HD therapy as it might allow specific inhibition of the mutant Htt gene expression and hence slow down disease progression. Support provided by uniQure and EU-FP7 E!7900.

Constitutive expression of short hairpin RNAs (shRNAs) may cause cellular toxicity in vivo and using microRNA (miRNA) scaffolds can circumvent this problem. Previously, we have shown that embedding small interfering RNA sequences targeting apolipoprotein B100 (ApoB) in shRNA (shApoB) or miRNA (miApoB) scaffolds resulted in differential processing and long-term efficacy in vivo. Here we show that adeno-associated virus (AAV)-shApoB- or AAV-miApoB-mediated ApoB knockdown induced differential liver morphology and transcriptome expression changes. Our analyses indicate that ApoB knockdown with both shApoB and miApoB resulted in alterations of genes involved in lipid metabolism. In addition, in AAV-shApoB-injected animals, genes involved in immune system activation or cell growth and death were affected, which was associated with increased hepatocyte proliferation. Subsequently, in AAV-miApoB-injected animals, changes of genes involved in oxidoreductase activity, oxidative phosphorylation and nucleic bases biosynthetic processes were observed. Our results demonstrate that long-term knockdown of ApoB in vivo by shApoB or miApoB induces several transcriptome changes in murine liver. The increased hepatocyte profileration by AAV-shRNA may have severe long-term effects indicating that AAV-mediated RNA interference therapy using artificial miRNA may be a safer approach for familial hypercholesterolemia therapy.

PURPOSE:The assessment of the capacity of bone marrow stromal cells (BMSC) to repair the nervous system using gene expression profiling. The evaluation of effects of long-term culturing on the gene expression profile of BMSC.METHODS:Fourty four k whole genome rat microarrays were used to study gene expression of cultured BMSC at passage (P)3 and to compare expression profiles between P3 and P14 BMSC. Quantitative PCR was employed to validate the microarray results.RESULTS:P3 BMSC expressed genes involved in neural developmental events such as glial differentiation, neuron proliferation, and neurite formation. They also express genes encoding for growth factors and for proteins involved in growth factor signaling. A total of 6687 genes were co-expressed in P3 and P14 BMSC. Of these co-expressed genes, 3% (202 genes) was differentially expressed with 159 genes higher in P3 BMSC and 43 genes higher in P14 BMSC. The gene expression patterns were independently validated using quantitative PCR. Functional data mining by Gene Ontology (GO)-analysis revealed that 85/159 and 22/43 genes were annotated in the GO database. In P3 BMSC, 53 GO-classes were overrepresented with several involved in organ development, cell proliferation, and neural repair. In P14 BMSC, three GO-classes were overrepresented with one involved in organ development.CONCLUSIONS:Our gene profiling results suggested a decreased plasticity and repair aptitude of long-term cultured BMSC. Our data indicated the use of early passage BMSC for neural repair approaches.

RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo.