Enhanced Factor IX Activity following Administration of AAV5-R338L "Padua" Factor IX versus AAV5 WT Human Factor IX in NHPs.

Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%-13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L "Padua" FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile.

Inhibition of Semaphorin3A Promotes Ocular Dominance Plasticity in the Adult Rat Visual Cortex.

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.

Optimization of viral protein ratios for production of rAAV serotype 5 in the baculovirus system.

Recombinant adeno-associated virus (rAAV) has become the vector of choice for the development of novel human gene therapies. High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-based system requires a genetic redesign of the viral protein 1 (VP1) operon. In this study, we generated a library of rationally designed rAAV serotype 5 variants with modulations in the translation-initiation region of VP1 and investigated the potency of the resulting vectors. We found that the initiation strength at the VP1 translational start had downstream effects on the VP2/VP3 ratio. Excessive incorporation of VP3 into a vector type decreased potency, even when the VP1/VP2 ratio was in balance. Finally, we successfully generated a potent rAAV vector based on serotype 5 with a balanced VP1/VP2/VP3 stoichiometry.

Small Scale Production of Recombinant Adeno-Associated Viral Vectors for Gene Delivery to the Nervous System.

Adeno-associated viral vectors have numerous applications in neuroscience, including the study of gene function in health and disease, targeting of light-sensitive proteins to anatomically distinct sets of neurons to manipulate neuronal activity (optogenetics), and the delivery of fluorescent protein to study anatomical connectivity in the brain. Moreover several phase I/II clinical trials for gene therapy of eye and brain diseases with adeno-associated viral vectors have shown that these vectors are well tolerated by human patients. In this chapter we describe a detailed protocol for the small scale production of recombinant adeno-associated viral vectors. This protocol can be executed by investigators with experience in cell culture and molecular biological techniques in any well-equipped molecular neurobiology laboratory. With this protocol we typically obtain research batches of 100-200 µL that range in titer from 5 × 1012 to 2 × 1013 genomic copies/mL.

Pathology associated with AAV mediated expression of beta amyloid or C100 in adult mouse hippocampus and cerebellum.

Accumulation of beta amyloid (Aß) in the brain is a primary feature of Alzheimer's disease (AD) but the exact molecular mechanisms by which Aß exerts its toxic actions are not yet entirely clear. We documented pathological changes 3 and 6 months after localised injection of recombinant, bi-cistronic adeno-associated viral vectors (rAAV2) expressing human Aß40-GFP, Aß42-GFP, C100-GFP or C100(V717F)-GFP into the hippocampus and cerebellum of 8 week old male mice. Injection of all rAAV2 vectors resulted in wide-spread transduction within the hippocampus and cerebellum, as shown by expression of transgene mRNA and GFP protein. Despite the lack of accumulation of Aß protein after injection with AAV vectors, injection of rAAV2-Aß42-GFP and rAAV2- C100(V717F)-GFP into the hippocampus resulted in significantly increased microgliosis and altered permeability of the blood brain barrier, the latter revealed by high levels of immunoglobulin G (IgG) around the injection site and the presence of IgG positive cells. In comparison, injection of rAAV2-Aß40-GFP and rAAV2-C100-GFP into the hippocampus resulted in substantially less neuropathology. Injection of rAAV2 vectors into the cerebellum resulted in similar types of pathological changes, but to a lesser degree. The use of viral vectors to express different types of Aß and C100 is a powerful technique with which to examine the direct in vivo consequences of Aß expression in different regions of the mature nervous system and will allow experimentation and analysis of pathological AD-like changes in a broader range of species other than mouse.

GFAP-driven GFP expression in activated mouse Müller glial cells aligning retinal blood vessels following intravitreal injection of AAV2/6 vectors.

BACKGROUND: Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy. METHODOLOGY/PRINCIPAL FINDINGS: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels. CONCLUSIONS/SIGNIFICANCE: Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.

Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system.

BACKGROUND: After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2). RESULTS: We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. CONCLUSIONS: RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.

Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.

For many experiments in the study of the peripheral nervous system, it would be useful to genetically manipulate primary sensory neurons. We have compared vectors based on adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, and 8, and lentivirus (LV), all expressing green fluorescent protein (GFP), for efficiency of transduction of sensory neurons, expression level, cellular tropism, and persistence of transgene expression following direct injection into the dorsal root ganglia (DRG), using histological quantification and qPCR. Two weeks after injection, AAV1, AAV5, and AAV6 had transduced the most neurons. The time course of GFP expression from these three vectors was studied from 1 to 12 weeks after injection. AAV5 was the most effective serotype overall, followed by AAV1. Both these serotypes showed increasing neuronal transduction rates at later time points, with some injections of AAV5 yielding over 90% of DRG neurons GFP(+) at 12 weeks. AAV6 performed well initially, but transduction rates declined dramatically between 4 and 12 weeks. AAV1 and AAV5 both transduced large-diameter neurons, IB4(+) neurons, and CGRP(+) neurons. In conclusion, AAV5 is a highly effective gene therapy vector for primary sensory neurons following direct injection into the DRG.

Adeno-associated viral vector (AAV)-mediated gene transfer in the red nucleus of the adult rat brain: comparative analysis of the transduction properties of seven AAV serotypes and lentiviral vectors.

Recombinant adeno-associated viral vectors (AAVs) are very promising gene transfer tools for the nervous system. We have compared the efficiency of gene expression of seven AAV serotypes in young adult rats following a single injection in a major nucleus of the mid brain, the red nucleus, which is the origin of the rubrospinal tract. AAV serotypes 1-6 and 8 and a lentiviral vector (LV) were used, all encoding green fluorescent protein (GFP) under control of the cytomegalovirus (CMV) promoter. AAV vectors were titer matched at 5x10(11) genomic copies (GC)/ml and 1mul was injected into the red nucleus. The proportion of transduced neurons in the red nucleus was determined at 1 and 4 weeks post-injection. AAV1 would be the vector of choice if the aim would be to overexpress a transgene at high level for a longer period of time. AAV5 and AAV8 would be the preferred serotype if onset of expression is should be somewhat delayed. The use of lentiviral vectors should be considered when transduction of both glial cells and neurons is required. Serotypes 3 and 4 did not transduce red nucleus neurons. AAV1, AAV6 and LV would be the vectors of choice if the aim of the experiment would be to rapidly express a transgene. The current data are important for the design of experiments that aim to study the effects of transgene products on the regenerative capacity of injured red nucleus neurons.

Human neuroma contains increased levels of semaphorin 3A, which surrounds nerve fibers and reduces neurite extension in vitro.

Neuroma formation after peripheral nerve injury is detrimental to functional recovery and is therefore a significant clinical problem. The molecular basis for this phenomenon is not fully understood. Here, we show that the expression of the chemorepulsive protein semaphorin 3A (sema3A), but not semaphorin 3F, is increased in human neuroma tissue that has formed in severe obstetric brachial plexus lesions. Sema3A is produced by fibroblasts in the epineurial space and appears to be secreted into the extracellular matrix. It surrounds fascicles, minifascicles, or single axons, suggesting a role in fasciculation and inhibition of neurite outgrowth. Lentiviral vector-mediated knock-down of Neuropilin 1, the receptor for sema3A, leads to increased neurite outgrowth of F11 cells cultured on neuroma tissue, but not of F11 cells cultured on normal nerve tissue. These findings demonstrate the putative inhibitory role of sema3A in human neuroma tissue. Our observations are the first demonstration of the expression of sema3A in human neural scar tissue and support a role for this protein in the inhibition of axonal regeneration in injured human peripheral nerves. These findings contribute to the understanding of the outgrowth inhibitory properties of neuroma tissue.

Presence of a transcriptionally active glucocorticoid receptor alpha in lens epithelial cells.

PURPOSE: The purpose of this study was to determine whether lens epithelial cells (LECs) contain a glucocorticoid receptor (GR) that is transcriptionally active and that is able to induce production of known glucocorticoid-inducible proteins. METHODS: Protein and mRNA were obtained from human, rabbit, and bovine lens epithelia and from cultured human lens epithelial cells (B3, hLECs) and rabbit lens epithelial cells (N/N1003A, rLECs). Paraffin-embedded sections were prepared from human lenses for immunohistochemical localization of GR. RT-PCR was performed to amplify portions of GR, and the products were sequenced. Protein samples were analyzed by Western blot. hLECs and rLECs were transfected with pTAT3-luc and assayed for luciferase activity after treatment with dexamethasone (Dex) and/or RU486. Dex-treated LECs were also analyzed by quantitative real-time PCR and by Western blot for expression of specific mRNA and proteins. RESULTS: By PCR and sequencing, products consistent with GR sequences were obtained from human, rabbit, and bovine lenses and from hLECs and rLECs. The complete GRalpha sequence was obtained from rLECs and was found to be 89% identical with human GR. A 1757-bp 3' fragment of bovine GRalpha cDNA was also amplified from bovine lens. By Western blot, bands of approximately 94 kDa, the expected size of GR, were identified from human, rabbit, and bovine lens samples and from hLECs and rLECs, using anti-GR antibodies. Anti-GR antisera localized GR to both the cytosol of anterior and bow region LECs and to the nuclei of epithelial and early-differentiating lens fiber cells. Luciferase expression was induced in pTAT3-luc-transfected rLECs and hLECs by Dex treatment and this expression was partially (rLECs) or completely (hLECs) blocked by pretreatment with RU486. mRNA levels for type-1 glucocorticoid-induced target genes and also mRNA and protein levels for type-2 genes were upregulated after Dex exposure. CONCLUSIONS: The data confirm the existence of GR in hLECs, indicate that GR is present in rLECs, and resolve the controversy over the presence of GR in bovine lens. The GRalpha in hLECs and rLECs was shown to be transcriptionally active and the expression levels in hLECs of mRNAs and proteins known to be regulated by glucocorticoids were modified in these cells by glucocorticoid treatment.