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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.
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Huntingtin (HTT)-lowering therapies hold promise to slow down neurodegeneration in Huntington's disease (HD). Here, we assessed the translatability and long-term durability of recombinant adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (rAAV5-miHTT) administered by magnetic resonance imaging-guided convention-enhanced delivery in transgenic HD minipigs. rAAV5-miHTT (1.2 × 1013 vector genome (VG) copies per brain) was successfully administered into the striatum (bilaterally in caudate and putamen), using age-matched untreated animals as controls. Widespread brain biodistribution of vector DNA was observed, with the highest concentration in target (striatal) regions, thalamus, and cortical regions. Vector DNA presence and transgene expression were similar at 6 and 12 months after administration. Expression of miHTT strongly correlated with vector DNA, with a corresponding reduction of mutant HTT (mHTT) protein of more than 75% in injected areas, and 30 to 50% lowering in distal regions. Translational pharmacokinetic and pharmacodynamic measures in cerebrospinal fluid (CSF) were largely in line with the effects observed in the brain. CSF miHTT expression was detected up to 12 months, with CSF mHTT protein lowering of 25 to 30% at 6 and 12 months after dosing. This study demonstrates widespread biodistribution, strong and durable efficiency of rAAV5-miHTT in disease-relevant regions in a large brain, and the potential of using CSF analysis to determine vector expression and efficacy in the clinic.
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Doença de Huntington , MicroRNAs , Animais , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/genética , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/terapia , MicroRNAs/metabolismo , Suínos , Porco Miniatura/metabolismo , Distribuição TecidualRESUMO
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.
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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.
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Envelhecimento/fisiologia , Dominância Ocular/fisiologia , Semaforina-3A/antagonistas & inibidores , Córtex Visual/fisiologia , Animais , Cones de Crescimento/metabolismo , Células HEK293 , Humanos , Neurônios/metabolismo , Neuropilinas/metabolismo , Agregados Proteicos , Ratos , Semaforina-3A/metabolismo , Solubilidade , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
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Expressão Gênica , Terapia Genética , Vetores Genéticos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Perda Auditiva/genética , Perda Auditiva/terapia , Neurônios/metabolismo , Animais , Biomarcadores , Cóclea/metabolismo , Cóclea/patologia , Modelos Animais de Doenças , Imunofluorescência , Técnicas de Transferência de Genes , Terapia Genética/métodos , Perda Auditiva/diagnóstico , Perda Auditiva/fisiopatologia , Humanos , Imuno-Histoquímica , Camundongos , FenótipoRESUMO
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.
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Terapia Genética/métodos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/terapia , Animais , Animais Geneticamente Modificados , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos/genética , Humanos , Doença de Huntington/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Suínos , Porco Miniatura , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Many therapeutic strategies aimed at relieving symptoms of Parkinson's 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.
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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.
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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.
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Convecção , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Putamen , Animais , Regulação da Expressão Gênica , Vetores Genéticos/genética , Infusões Intraventriculares , Macaca mulatta , Masculino , Putamen/cirurgiaRESUMO
Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5' and 3' cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 10(11) genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.
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Apolipoproteína B-100/genética , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Western Blotting , Colesterol/sangue , Técnicas de Silenciamento de Genes , Vetores Genéticos , Células HEK293 , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/química , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/químicaRESUMO
PURPOSE: To evaluate the ability of mesenchymal stem cells (MSCs) engineered to produce and secrete brain-derived neurotrophic factor (BDNF) to protect retinal function and structure after intravitreal transplantation in a rat model of chronic ocular hypertension (COH). METHODS: COH was induced by laser cauterization of trabecular meshwork and episcleral veins in rat eyes. COH eyes received an intravitreal transplant of MSCs engineered to express BDNF and green fluorescent protein (BDNF-MSCs) or just GFP (GFP-MSCs). Computerized pupillometry and electroretinography (ERG) were performed to assess optic nerve and retinal function. Quantification of optic nerve damage was performed by counting retinal ganglion cells (RGCs) and evaluating optic nerve cross-sections. RESULTS: After transplantation into COH eyes, BDNF-MSCs preserved significantly more retina and optic nerve function than GFP-MSC-treated eyes when pupil light reflex (PLR) and ERG function were evaluated. PLR analysis showed significantly better function (P = 0.03) in BDNF-MSC-treated eyes (operated/control ratio = 63.00% ± 11.39%) than GFP-MSC-treated eyes (operated/control ratio = 31.81% ± 9.63%) at 42 days after surgery. The BDNF-MSC-transplanted eyes also displayed a greater level of RGC preservation than eyes that received the GFP-MSCs only (RGC cell counts: BDNF-MSC-treated COH eyes, 112.2 ± 19.39 cells/section; GFP-MSC-treated COH eyes, 52.21 ± 11.54 cells/section; P = 0.01). CONCLUSIONS: The authors have demonstrated that lentiviral-transduced BDNF-producing MSCs can survive in eyes with chronic hypertension and can provide retina and optic nerve functional and structural protection. Transplantation of BDNF-producing stem cells may be a viable treatment strategy for glaucoma.
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Fator Neurotrófico Derivado do Encéfalo/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Hipertensão Ocular/cirurgia , Doenças do Nervo Óptico/prevenção & controle , Nervo Óptico/fisiopatologia , Corpo Vítreo/cirurgia , Animais , Doença Crônica , Modelos Animais de Doenças , Eletrorretinografia , Células-Tronco Mesenquimais/citologia , Hipertensão Ocular/complicações , Hipertensão Ocular/fisiopatologia , Nervo Óptico/patologia , Doenças do Nervo Óptico/etiologia , Ratos , Ratos Endogâmicos BN , Retina/patologia , Retina/fisiopatologia , Resultado do TratamentoRESUMO
Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels in mice, RNA interference (RNAi) was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs (shRNAs) targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed our focus to one candidate for in vivo testing. Self-complementary adeno-associated virus serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB. A strong dose-dependent knockdown of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. Furthermore, shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, elevated lipid accumulation was shown in murine liver transduced with shApoB, a known phenotypic side effect of lowering ApoB levels. These results demonstrate a robust dose-dependent knockdown of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAi-based gene therapy for the treatment of hypercholesterolemia.
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Apolipoproteínas B/genética , Colesterol/sangue , Dependovirus/genética , Vetores Genéticos/genética , RNA Interferente Pequeno/genética , Animais , Apolipoproteínas B/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Colesterol/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
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Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Neuroglia/metabolismo , Vasos Retinianos/citologia , Transdução Genética/métodos , Adenoviridae/genética , Animais , Expressão Gênica , Humanos , Injeções , Camundongos , Regiões Promotoras Genéticas/genética , Transgenes/genéticaRESUMO
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.
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Dependovirus/classificação , Gânglios Espinais , Terapia Genética , Vetores Genéticos , Animais , Dependovirus/genética , Feminino , Plasmídeos , Ratos , Ratos Wistar , Sorotipagem , Transdução GenéticaRESUMO
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.
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Dependovirus/classificação , Dependovirus/genética , Expressão Gênica/genética , Vetores Genéticos , Núcleo Rubro/metabolismo , Transdução Genética/métodos , Animais , Feminino , Engenharia Genética , Vetores Genéticos/classificação , Proteínas de Fluorescência Verde/genética , Ratos , Ratos Endogâmicos F344 , Medula Espinal/metabolismoRESUMO
The purpose of this study was to determine the viability of cell-based delivery of brain-derived neurotrophic factor (BDNF) from genetically modified mesenchymal stem cells (MSCs) for neuroprotection of RGC-5 cells. RGC-5 cells were differentiated with the protein kinase inhibitor staurosporine (SS) and exposed to the cellular stressors glutamate or H2O2. As a neuroprotective strategy, these cells were then co-cultured across a membrane insert with mesenchymal stem cells (MSCs) engineered with a lentiviral vector for production of BDNF (BDNF-MSCs). As a positive control, recombinant human BDNF (rhBDNF) was added to stressed RGC-5 cells. After SS-differentiation RGC-5s developed neuronal-like morphologies, and a significant increase in the proportion of RGC-5s immunoreactive for TuJ-1 and Brn3a was observed. Differentiated RGC-5s also had prominent TrkB staining, demonstrating expression of the high-affinity BDNF receptor. Treatment of SS-differentiated RGC-5s with glutamate or H2O2, produced significant cell death (56.0 +/- 7.02 and 48.90 +/- 4.58% of control cells, respectively) compared to carrier-solution treated cells. BDNF-delivery from MSCs preserved more RGC-5 cells after treatment with glutamate (80.0 +/- 5.40% cells remaining) than control GFP expressing MSCs (GFP-MSCs, 57.29 +/- 1.89%, p < 0.01). BDNF-MSCs also protected more RGC-5s after treatment with H2O2 (65.6 +/- 3.47%) than GFP-MSCs (46.0 +/- 4.20%, p < 0.01). We have shown survival of differentiated RGC-5s is reduced by the cellular stressors glutamate and H2O2. Additionally, our results demonstrate that genetically modified BDNF-producing MSCs can enhance survival of stressed RGC-5 cells and therefore, may be effective vehicles to deliver BDNF to retinal ganglion cells affected by disease.
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Fator Neurotrófico Derivado do Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Ácido Glutâmico/toxicidade , Peróxido de Hidrogênio/toxicidade , Células-Tronco Mesenquimais/metabolismo , Células Ganglionares da Retina/citologia , Estaurosporina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Engenharia Genética , Fármacos Neuroprotetores/farmacologia , Ratos , Receptor trkB/metabolismo , Proteínas Recombinantes/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3A/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Damaged CNS axons are prevented from regenerating by an environment containing many inhibitory factors. They also lack an integrin that interacts with tenascin-C, the main extracellular matrix glycoprotein of the CNS, which is upregulated after injury. The alpha9beta1 integrin heterodimer is a receptor for the nonalternatively spliced region of tenascin-C, but the alpha9 subunit is absent in adult neurons. In this study, we show that PC12 cells and adult rat dorsal root ganglion (DRG) neurons do not extend neurites on tenascin-C. However, after forced expression of alpha9 integrin, extensive neurite outgrowth from PC12 cells and adult rat DRG neurons occurs. Moreover, both DRG neurons and PC12 cells secrete tenascin-C, enabling alpha9-transfected cells to grow axons on tissue culture plastic. Using adeno-associated viruses to express alpha9 integrin in vivo in DRGs, we examined axonal regeneration after cervical dorsal rhizotomy or dorsal column crush in the adult rat. After rhizotomy, significantly more dorsal root axons regrew into the dorsal root entry zone at 6 weeks after injury in alpha9 integrin-expressing animals than in green fluorescent protein (GFP) controls. Similarly, after a dorsal column crush injury, there was significantly more axonal growth into the lesion site compared with GFP controls at 6 weeks after injury. Behavioral analysis after spinal cord injury revealed that both experimental and control groups had an increased withdrawal latency in response to mechanical stimulation when compared with sham controls; however, in response to heat stimulation, normal withdrawal latencies returned after alpha9 integrin treatment but remained elevated in control groups.
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Axônios/metabolismo , Cadeias alfa de Integrinas/fisiologia , Regeneração Nervosa/fisiologia , Neuritos/fisiologia , Células Receptoras Sensoriais/metabolismo , Tenascina/metabolismo , Animais , Axônios/ultraestrutura , Células Cultivadas , Gânglios Espinais/fisiologia , Gânglios Espinais/ultraestrutura , Humanos , Cadeias alfa de Integrinas/biossíntese , Cadeias alfa de Integrinas/genética , Masculino , Microscopia Confocal , Neuritos/ultraestrutura , Neurogênese/fisiologia , Células PC12 , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/fisiologia , Células Receptoras Sensoriais/ultraestruturaRESUMO
We sought to directly compare growth and myelination of local and supraspinal axons by implanting into the injured spinal cord Schwann cells (SCs) transduced ex vivo with adenoviral (AdV) or lentiviral (LV) vectors encoding a bifunctional neurotrophin molecule (D15A). D15A mimics actions of both neurotrophin-3 and brain-derived neurotrophic factor. Transduced SCs were injected into the injury center 1 week after a moderate thoracic (T8) adult rat spinal cord contusion. D15A expression and bioactivity in vitro; D15A levels in vivo; and graft volume, SC number, implant axon number and cortico-, reticulo-, raphe-, coerulo-spinal and sensory axon growth were determined for both types of vectors employed to transduce SCs. ELISAs revealed that D15A-secreting SC implants contained significantly higher levels of neurotrophin than non-transduced SC and AdV/GFP and LV/GFP SC controls early after implantation. At 6 weeks post-implantation, D15A-secreting SC grafts exhibited 5-fold increases in graft volume, SC number and myelinated axon counts and a 3-fold increase in myelinated to unmyelinated (ensheathed) axon ratios. The total number of axons within grafts of LV/GFP/D15A SCs was estimated to be over 70,000. Also 5-HT, DbetaH, and CGRP axon length was increased up to 5-fold within D15A grafts. In sum, despite qualitative differences using the two vectors, increased neurotrophin secretion by the implanted D15A SCs led to the presence of a significantly increased number of axons in the contusion site. These results demonstrate the therapeutic potential for utilizing neurotrophin-transduced SCs to repair the injured spinal cord.
Assuntos
Axônios/fisiologia , Transplante de Células , Fatores de Crescimento Neural/metabolismo , Células de Schwann/fisiologia , Traumatismos da Medula Espinal/cirurgia , Transdução Genética/métodos , Análise de Variância , Animais , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Comportamento Animal , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Transplante de Células/métodos , Dopamina beta-Hidroxilase/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Regeneração Nervosa/fisiologia , Ratos , Ratos Endogâmicos F344 , Nervo Isquiático/citologia , Serotonina/metabolismoRESUMO
For regrowth of injured nerve fibers following spinal cord injury (SCI), the environment must be favorable for axonal growth. The delivery of a therapeutic gene, beneficial for axonal growth, into the central nervous system for repair can be accomplished in many ways. Perhaps the most simple and elegant strategy is the so-called direct gene therapy approach that uses a single injection for delivery of a gene therapy vehicle. Among the vectors that have been used to transduce neural tissue in vivo are non-viral, herpes simplex viral, adeno-associated viral, adenoviral, and lentiviral vectors, each with their own merits and limitations. Many studies have been undertaken using direct gene therapy, ranging from strategies for neuroprotection to axonal growth promotion at the injury site, dorsal root injury repair, and initiation of a growth-supporting genetic program. The limitations and successes of direct gene transfer for spinal cord repair are discussed in this review.
Assuntos
Terapia Genética , Traumatismos da Medula Espinal/terapia , Animais , Vetores Genéticos , Humanos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/uso terapêuticoRESUMO
Freeze-dried poly(D,L-lactic acid) macroporous scaffold filled with a fibrin solution containing Schwann cells (SCs) lentivirally transduced to produce and secrete D15A, a bi-functional neurotrophin with brain-derived neurotrophic factor and neurotrophin-3 activity, and to express green fluorescent protein (GFP) were implanted in the completely transected adult rat thoracic spinal cord. Control rats were similarly injured and then implanted with scaffolds containing the fibrin solution with SCs lentivirally transduced to produce express GFP only or with the fibrin solution only. Transgene production and biological activity in vitro, SC survival within the scaffold in vitro and in vivo, scaffold integration, axonal regeneration and myelination, and hind limb motor function were analyzed at 1, 2, and 6 weeks after implantation. In vitro, lentivirally transduced SCs produced 87.5 ng/24 h/10(6) cells of D15A as measured by neurotrophin-3 activity in ELISA. The secreted D15A was biologically active as evidenced by its promotion of neurite outgrowth of dorsal root ganglion neurons in culture. In vitro, SCs expressing GFP were present in the scaffolds for up to 6 h, the end of a typical surgery session. Implantation of SC-seeded scaffolds caused modest loss of spinal nervous tissue. Reactive astrocytes and chondroitin sulfate glycosaminoglycans were present in spinal tissue adjacent to the scaffold. Vascularization of the scaffold was ongoing at 1 week post-implantation. There were no apparent differences in scaffold integration and blood vessel formation between groups. A decreasing number of implanted (GFP-positive) SCs were found within the scaffold during the first 3 days after implantation. Apoptosis was identified as one of the mechanisms of cell death. At 1 week and later time points after implantation, few of the implanted SCs were present in the scaffold. Neurofilament-positive axons were found in the scaffold. At 6 weeks post-grafting, myelinated axons were observed within and at the external surface of the scaffold. Axons did not grow from the scaffold into the caudal cord. All groups demonstrated a similar improvement of hind limb motor function. Our findings demonstrated that few seeded SCs survived in vivo, which could account for the modest axonal regeneration response into and across the scaffold. For the development of SC-seeded macroporous scaffolds that effectively promote axonal regeneration in the injured spinal cord, the survival and/or total number of SCs in the scaffold needs to be improved.