RESUMO
Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.
Assuntos
Dependovirus , Vetores Genéticos , Transfecção , Humanos , Dependovirus/genética , Dependovirus/isolamento & purificação , Células HEK293 , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Transfecção/métodos , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Polietilenoimina/química , Reatores Biológicos , Cromatografia por Troca Iônica/métodos , Vírion/genética , Vírion/isolamento & purificaçãoRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by motor neuron loss and skeletal muscle atrophy. SMA is caused by the loss of the SMN1 gene and low SMN protein levels. Current SMA therapies work by increasing SMN protein in the body. Although SMA is regarded as a motor neuron disorder, growing evidence shows that several peripheral organs contribute to SMA pathology. A gene therapy treatment, onasemnogene abeparvovec, is being explored in clinical trials via both systemic and central nervous system (CNS) specific delivery, but the ideal route of delivery as well as the long-term effectiveness is unclear. To investigate the impact of gene therapy long term, we assessed SMA mice at 6 months after treatment of either intravenous (IV) or intracerebroventricular (ICV) delivery of scAAV9-cba-SMN. Interestingly, we observed that SMN protein levels were restored in the peripheral tissues but not in the spinal cord at 6 months of age. However, ICV injections provided better motor neuron and motor function protection than IV injection, while IV-injected mice demonstrated better protection of neuromuscular junctions and muscle fiber size. Surprisingly, both delivery routes resulted in an equal rescue on survival, weight, and liver and pancreatic defects. These results demonstrate that continued peripheral AAV9-SMN gene therapy is beneficial for disease improvement even in the absence of SMN restoration in the spinal cord.
Assuntos
Atrofia Muscular Espinal , Animais , Camundongos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Neurônios Motores , Modelos Animais de Doenças , Sistema Nervoso Central , Terapia GenéticaRESUMO
The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrPC (GDL) can also initiate neurotoxicity by inducing an intramolecular R208 -H140 hydrogen bond ("H-latch") between the α2-α3 and ß2-α2 loops of PrPC . Importantly, GDL that suppresses the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced 19 individual PrPC variants to PrPC -deficient cerebellar organotypic cultured slices using adenovirus-associated viral vectors (AAV). We report that GDL toxicity requires a single N-proximal cationic residue (K27 or R27 ) within PrPC . Alanine substitution of K27 also prevented the toxicity of PrPC mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co-expression of wild-type PrPC . K27 may represent an actionable target for compounds aimed at preventing prion-related neurodegeneration.
Assuntos
Doenças Neurodegenerativas , Doenças Priônicas , Príons , Camundongos , Animais , Proteínas Priônicas/genética , Genética Reversa , Príons/genética , Anticorpos , Doenças Priônicas/genéticaRESUMO
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the SMN1 gene and low SMN protein levels. Although lower motor neurons are a primary target, there is evidence that peripheral organ defects contribute to SMA. Current SMA gene therapy and clinical trials use a single intravenous bolus of the blood-brain-barrier penetrant scAAV9-cba-SMN by either systemic or central nervous system (CNS) delivery, resulting in impressive amelioration of the clinical phenotype but not a complete cure. The impact of scAAV9-cba-SMN treatment regimens on the CNS as well as on specific peripheral organs is yet to be described in a comparative manner. Therefore, we injected SMA mice with scAAV9-cba-SMN either intravenously (IV) for peripheral SMN restoration or intracerebroventricularly (ICV) for CNS-focused SMN restoration. In our system, ICV injections increased SMN in peripheral organs and the CNS while IV administration increased SMN in peripheral tissues only, largely omitting the CNS. Both treatments rescued several peripheral phenotypes while only ICV injections were neuroprotective. Surprisingly, both delivery routes resulted in a robust rescue effect on survival, weight, and motor function, which in IV-treated mice relied on peripheral SMN restoration but not on targeting the motor neurons. This demonstrates the independent contribution of peripheral organs to SMA pathology and suggests that treatments should not be restricted to motor neurons.
Assuntos
Dependovirus , Atrofia Muscular Espinal , Animais , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/genética , Camundongos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/terapia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Skeletal muscle displays remarkable plasticity upon exercise and is also one of the organs most affected by aging. Despite robust evidence that aging is associated with loss of fast-twitch (type II) muscle fibers, the underlying mechanisms remain to be elucidated. Here, we identified an exercise-induced long noncoding RNA, CYTOR, whose exercise responsiveness was conserved in human and rodents. Cytor overexpression in mouse myogenic progenitor cells enhanced myogenic differentiation by promoting fast-twitch cell fate, whereas Cytor knockdown deteriorated expression of mature type II myotubes. Skeletal muscle Cytor expression was reduced upon mouse aging, and Cytor expression in young mice was required to maintain proper muscle morphology and function. In aged mice, rescuing endogenous Cytor expression using adeno-associated virus serotype 9 delivery of CRISPRa reversed the age-related decrease in type II fibers and improved muscle mass and function. In humans, CYTOR expression correlated with type II isoform expression and was decreased in aged myoblasts. Increased CYTOR expression, mediated by a causal cisexpression quantitative trait locus located within a CYTOR skeletal muscle enhancer element, was associated with improved 6-min walk performance in aged individuals from the Helsinki Birth Cohort Study. Direct CYTOR overexpression using CRISPRa in aged human donor myoblasts enhanced expression of type II myosin isoforms. Mechanistically, Cytor reduced chromatin accessibility and occupancy at binding motifs of the transcription factor Tead1 by binding, and hence sequestering, Tead1. In conclusion, the long noncoding RNA Cytor was found to be a regulator of fast-twitch myogenesis in aging.
Assuntos
RNA Longo não Codificante , Envelhecimento/genética , Animais , Diferenciação Celular/genética , Estudos de Coortes , Humanos , Camundongos , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
α-Synuclein aggregation at the synapse is an early event in Parkinson's disease and is associated with impaired striatal synaptic function and dopaminergic neuronal death. The cysteine string protein (CSPα) and α-synuclein have partially overlapping roles in maintaining synaptic function and mutations in each cause neurodegenerative diseases. CSPα is a member of the DNAJ/HSP40 family of co-chaperones and like α-synuclein, chaperones the SNARE complex assembly and controls neurotransmitter release. α-Synuclein can rescue neurodegeneration in CSPαKO mice. However, whether α-synuclein aggregation alters CSPα expression and function is unknown. Here we show that α-synuclein aggregation at the synapse is associated with a decrease in synaptic CSPα and a reduction in the complexes that CSPα forms with HSC70 and STGa. We further show that viral delivery of CSPα rescues in vitro the impaired vesicle recycling in PC12 cells with α-synuclein aggregates and in vivo reduces synaptic α-synuclein aggregates increasing monomeric α-synuclein and restoring normal dopamine release in 1-120hαSyn mice. These novel findings reveal a mechanism by which α-synuclein aggregation alters CSPα at the synapse, and show that CSPα rescues α-synuclein aggregation-related phenotype in 1-120hαSyn mice similar to the effect of α-synuclein in CSPαKO mice. These results implicate CSPα as a potential therapeutic target for the treatment of early-stage Parkinson's disease.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Agregação Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Animais , Corpo Estriado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/patologia , Sinapses/metabolismo , Sinapses/patologiaRESUMO
AAV-mediated gene therapy is a promising approach for treating genetic hearing loss. Replacement or editing of the Tmc1 gene, encoding hair cell mechanosensory ion channels, is effective for hearing restoration in mice with some limitations. Efficient rescue of outer hair cell function and lack of hearing recovery with later-stage treatment remain issues to be solved. Exogenous genes delivered with the adeno-associated virus (AAV)9-PHP.B capsid via the utricle transduce both inner and outer hair cells of the mouse cochlea with high efficacy. Here, we demonstrate that AAV9-PHP.B gene therapy can promote hair cell survival and successfully rescues hearing in three distinct mouse models of hearing loss. Tmc1 replacement with AAV9-PHP.B in a Tmc1 knockout mouse rescues hearing and promotes hair cell survival with equal efficacy in inner and outer hair cells. The same treatment in a recessive Tmc1 hearing-loss model, Baringo, partially recovers hearing even with later-stage treatment. Finally, dual delivery of Streptococcus pyogenes Cas9 (SpCas9) and guide RNA (gRNA) in separate AAV9-PHP.B vectors selectively disrupts a dominant Tmc1 allele and preserves hearing in Beethoven mice, a model of dominant, progressive hearing loss. Tmc1-targeted gene therapies using single or dual AAV9-PHP.B vectors offer potent and versatile approaches for treating dominant and recessive deafness.
Assuntos
Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Perda Auditiva/terapia , Proteínas de Membrana/fisiologia , RNA Guia de Cinetoplastídeos/genética , Animais , Feminino , Vetores Genéticos/genética , Perda Auditiva/genética , Perda Auditiva/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Viral delivery of exogenous coding sequences into the inner ear has the potential for therapeutic benefit for patients suffering genetic or acquired hearing loss. To devise improved strategies for viral delivery, we investigated two injection techniques, round window membrane injection or a novel utricle injection method, for their ability to safely and efficiently transduce sensory hair cells and neurons of the mouse inner ear. In addition, we evaluated three synthetic AAV vectors (Anc80L65, AAV9-PHP.B, AAV2.7m8) encoding enhanced green fluorescent protein (eGFP) and three promoters (Cmv, Synapsin, Gfap) for their ability to transduce and drive expression in desired cell types. We found the utricle injection method with AAV9-PHP.B and a Cmv promoter was the most efficient combination for driving robust eGFP expression in both inner and outer hair cells. We found eGFP expression levels rose over 3-5 days post-injection, a viral dose of 1.5 × 109 gc yielded half maximal eGFP expression and that the utricle injection method yielded transduced hair cells even when delivered as late as postnatal day 16. Sensory transduction and auditory thresholds were unaltered in injected mice relative to uninjected wild-type controls. Vestibular end organs were also transduced without affecting balance behavior. The Synapsin promoter and the Gfap promoter drove strong eGFP expression in inner ear neurons and supporting cells, respectively. We conclude the AAV9-PHP.B vector and the utricle injection method are well-suited for delivery of exogenous gene constructs into inner ears of mouse models of auditory and vestibular dysfunction.
Assuntos
Orelha Interna , Animais , Infecções por Citomegalovirus , Dependovirus/genética , Terapia Genética , Vetores Genéticos , Células Ciliadas Auditivas Externas , Camundongos , Sáculo e Utrículo , Sinapsinas/genéticaRESUMO
Since most dominant human mutations are single nucleotide substitutions1,2, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve3 because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA3,4. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice5, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells6. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.
Assuntos
Alelos , Edição de Genes , Perda Auditiva Neurossensorial/prevenção & controle , Proteínas de Membrana/genética , Animais , Proteína 9 Associada à CRISPR/fisiologia , Linhagem Celular , Células Cultivadas , Dependovirus/genética , Modelos Animais de Doenças , Perda Auditiva Neurossensorial/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.
RESUMO
Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Cromatografia de Afinidade , Dependovirus/isolamento & purificação , Terapia Genética , Vetores Genéticos/isolamento & purificação , Células HEK293 , HumanosRESUMO
Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among the elderly. To understand its pathogenesis and to test therapies, animal models that faithfully reproduce key pathological PD hallmarks are needed. As a prelude to developing a model of PD, we tested the tropism, efficacy, biodistribution, and transcriptional effect of canine adenovirus type 2 (CAV-2) vectors in the brain of Microcebus murinus, a nonhuman primate that naturally develops neurodegenerative lesions. We show that introducing helper-dependent (HD) CAV-2 vectors results in long-term, neuron-specific expression at the injection site and in afferent nuclei. Although HD CAV-2 vector injection induced a modest transcriptional response, no significant adaptive immune response was generated. We then generated and tested HD CAV-2 vectors expressing leucine-rich repeat kinase 2 (LRRK2) and LRRK2 carrying a G2019S mutation (LRRK2G2019S), which is linked to sporadic and familial autosomal dominant forms of PD. We show that HD-LRRK2G2019S expression induced parkinsonian-like motor symptoms and histological features in less than 4 months.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/farmacologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Adenovirus Caninos/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Cheirogaleidae , Feminino , Perfilação da Expressão Gênica , Vetores Genéticos , Masculino , Mutação , Neurônios/efeitos dos fármacos , Técnicas Estereotáxicas , Distribuição Tecidual , Transcriptoma , Transdução Genética , TropismoRESUMO
Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are the two most common motoneuron disorders, which share typical pathological hallmarks while remaining genetically distinct. Indeed, SMA is caused by deletions or mutations in the survival motor neuron 1 (SMN1) gene whilst ALS, albeit being mostly sporadic, can also be caused by mutations within genes, including superoxide dismutase 1 (SOD1), Fused in Sarcoma (FUS), TAR DNA-binding protein 43 (TDP-43) and chromosome 9 open reading frame 72 (C9ORF72). However, it has come to light that these two diseases may be more interlinked than previously thought. Indeed, it has recently been found that FUS directly interacts with an Smn-containing complex, mutant SOD1 perturbs Smn localization, Smn depletion aggravates disease progression of ALS mice, overexpression of SMN in ALS mice significantly improves their phenotype and lifespan, and duplications of SMN1 have been linked to sporadic ALS. Beyond genetic interactions, accumulating evidence further suggests that both diseases share common pathological identities such as intrinsic muscle defects, neuroinflammation, immune organ dysfunction, metabolic perturbations, defects in neuron excitability and selective motoneuron vulnerability. Identifying common molecular effectors that mediate shared pathologies in SMA and ALS would allow for the development of therapeutic strategies and targeted gene therapies that could potentially alleviate symptoms and be equally beneficial in both disorders. In the present review, we will examine our current knowledge of pathogenic commonalities between SMA and ALS, and discuss how furthering this understanding can lead to the establishment of novel therapeutic approaches with wide-reaching impact on multiple motoneuron diseases.
Assuntos
Esclerose Lateral Amiotrófica/genética , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/terapia , Animais , Proteína C9orf72/genética , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/terapia , Proteína FUS de Ligação a RNA/genética , Superóxido Dismutase-1/genéticaRESUMO
α-Synuclein misfolding and aggregation is often accompanied by ß-amyloid deposition in some neurodegenerative diseases. We hypothesised that α-synuclein promotes ß-amyloid production from APP. ß-Amyloid levels and APP amyloidogenic processing were investigated in neuronal cell lines stably overexpressing wildtype and mutant α-synuclein. γ-Secretase activity and ß-secretase expression were also measured. We show that α-synuclein expression induces ß-amyloid secretion and amyloidogenic processing of APP in neuronal cell lines. Certain mutations of α-synuclein potentiate APP amyloidogenic processing. γ-Secretase activity was not enhanced by wildtype α-synuclein expression, however ß-secretase protein levels were induced. Furthermore, a correlation between α-synuclein and ß-secretase protein was seen in rat brain striata. Iron chelation abolishes the effect of α-synuclein on neuronal cell ß-amyloid secretion, whereas overexpression of the ferrireductase enzyme Steap3 is robustly pro-amyloidogenic. We propose that α-synuclein promotes ß-amyloid formation by modulating ß-cleavage of APP, and that this is potentially mediated by the levels of reduced iron and oxidative stress.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , alfa-Sinucleína/metabolismo , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Feminino , Humanos , Ferro/metabolismo , Mutação , Neurônios/metabolismo , Proteínas Oncogênicas/metabolismo , Estresse Oxidativo , Oxirredutases , Proteólise , Ratos , Ratos Sprague-Dawley , alfa-Sinucleína/genéticaRESUMO
Neuromodulation by spinal cord stimulation has been proposed as a symptomatic treatment for Parkinson's disease. We tested the chronic effects of spinal cord stimulation in a progressive model of Parkinson's based on overexpression of alpha-synuclein in the substantia nigra. Adult Sprague Dawley rats received unilateral injections of adeno-associated virus serotype 6 (AAV6) in the substantia nigra to express alpha-synuclein. Locomotion and forepaw use of the rats were evaluated during the next 10 weeks. Starting on week 6, a group of AAV6-injected rats received spinal cord stimulation once a week. At the end of the experiment, tyrosine hydroxylase and alpha-synuclein immunostaining were performed. Rats with unilateral alpha-synuclein expression showed a significant decrease in the use of the contralateral forepaw, which was mildly but significantly reverted by spinal cord stimulation applied once a week from the 6th to the 10th week after the AAV6 injection. Long-term spinal cord stimulation proved to be effective to suppress or delay motor symptoms in a sustained and progressive model of Parkinson's and might become an alternative, less invasive neuromodulation option to treat this disease.
Assuntos
Comportamento Animal/fisiologia , Doença de Parkinson/terapia , Estimulação da Medula Espinal/métodos , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMO
The implantation of genetically modified cells is considered for the chronic delivery of therapeutic recombinant proteins in vivo. In the context of gene therapy, the genetic engineering of cells faces two main challenges. First, it is critical to generate expandable cell sources, which can maintain stable high productivity of the recombinant protein of interest over time, both in culture and after transplantation. In addition, gene transfer techniques need to be developed to engineer cells synthetizing complex polypeptides, such as recombinant monoclonal antibodies, to broaden the range of potential therapeutic applications. Here, we provide a workflow for the use of lentiviral vectors as a flexible tool to generate antibody-producing cells. In particular, lentiviral vectors can be used to genetically engineer the cell types compatible with encapsulation devices protecting the implanted cells from the host immune system. Detailed methods are provided for the design and production of lentiviral vectors, optimization of cell transduction, as well as for the quantification and quality control of the produced recombinant antibody.
Assuntos
Células Produtoras de Anticorpos/imunologia , Lentivirus/genética , Proteínas Recombinantes/genética , Transdução Genética/métodos , Células Produtoras de Anticorpos/metabolismo , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Vetores Genéticos , Humanos , Proteínas Recombinantes/imunologiaRESUMO
Genetic hearing loss accounts for up to 50% of prelingual deafness worldwide, yet there are no biologic treatments currently available. To investigate gene therapy as a potential biologic strategy for restoration of auditory function in patients with genetic hearing loss, we tested a gene augmentation approach in mouse models of genetic deafness. We focused on DFNB7/11 and DFNA36, which are autosomal recessive and dominant deafnesses, respectively, caused by mutations in transmembrane channel-like 1 (TMC1). Mice that carry targeted deletion of Tmc1 or a dominant Tmc1 point mutation, known as Beethoven, are good models for human DFNB7/11 and DFNA36. We screened several adeno-associated viral (AAV) serotypes and promoters and identified AAV2/1 and the chicken ß-actin (Cba) promoter as an efficient combination for driving the expression of exogenous Tmc1 in inner hair cells in vivo. Exogenous Tmc1 or its closely related ortholog, Tmc2, were capable of restoring sensory transduction, auditory brainstem responses, and acoustic startle reflexes in otherwise deaf mice, suggesting that gene augmentation with Tmc1 or Tmc2 is well suited for further development as a strategy for restoration of auditory function in deaf patients who carry TMC1 mutations.
Assuntos
Surdez/terapia , Terapia Genética , Audição , Proteínas de Membrana/genética , Animais , Surdez/genética , Dependovirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos MutantesRESUMO
Ex vivo gene therapy using retrievable encapsulated cellular implants is an effective strategy for the local and/or chronic delivery of therapeutic proteins. In particular, it is considered an innovative approach to modulate the activity of the immune system. Two recently proposed therapeutic schemes using genetically engineered encapsulated cells are discussed here: the chronic administration of monoclonal antibodies for passive immunization against neurodegenerative diseases and the local delivery of a cytokine as an adjuvant for anti-cancer vaccines.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Imunoterapia/métodos , Neoplasias/terapia , Doenças Neurodegenerativas/terapia , Adjuvantes Imunológicos/administração & dosagem , Doença de Alzheimer/imunologia , Doença de Alzheimer/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Citocinas/administração & dosagem , Engenharia Genética/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Humanos , Sistema Imunitário , Imunização Passiva/métodos , Neoplasias/imunologia , Doenças Neurodegenerativas/imunologia , Proteínas Recombinantes/administração & dosagemRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). LRRK2 contains functional GTPase and kinase domains. The most common G2019S mutation enhances the kinase activity of LRRK2 in vitro whereas G2019S LRRK2 expression in cultured neurons induces toxicity in a kinase-dependent manner. These observations suggest a potential role for kinase activity in LRRK2-associated PD. We have recently developed a novel rodent model of PD with progressive neurodegeneration induced by the adenoviral-mediated expression of G2019S LRRK2. In the present study, we further characterize this LRRK2 model and determine the contribution of kinase activity to LRRK2-mediated neurodegeneration. Recombinant human adenoviral vectors were employed to deliver human wild-type, G2019S or kinase-inactive G2019S/D1994N LRRK2 to the rat striatum. LRRK2-dependent pathology was assessed in the striatum, a region where LRRK2 protein is normally enriched in the mammalian brain. Human LRRK2 variants are robustly expressed throughout the rat striatum. Expression of G2019S LRRK2 selectively induces the accumulation of neuronal ubiquitin-positive inclusions accompanied by neurite degeneration and the altered distribution of axonal phosphorylated neurofilaments. Importantly, the introduction of a kinase-inactive mutation (G2019S/D1994N) completely ameliorates the pathological effects of G2019S LRRK2 in the striatum supporting a kinase activity-dependent mechanism for this PD-associated mutation. Collectively, our study further elucidates the pathological effects of the G2019S mutation in the mammalian brain and supports the development of kinase inhibitors as a potential therapeutic approach for treating LRRK2-associated PD. This adenoviral rodent model provides an important tool for elucidating the molecular basis of LRRK2-mediated neurodegeneration.
Assuntos
Adenoviridae/fisiologia , Corpo Estriado/patologia , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Feminino , Membro Anterior/fisiopatologia , Regulação da Expressão Gênica/genética , Glicina/genética , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson/fisiopatologia , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Wistar , Serina/genética , Fatores de Tempo , Transdução Genética , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Mechanisms underlying motor neuron subtype-selective endoplasmic reticulum (ER) stress and associated axonal pathology in amyotrophic lateral sclerosis (ALS) remain unclear. Here we show that the molecular environment of the ER between motor neuron subtypes is distinct, with characteristic signatures. We identify cochaperone SIL1, mutated in Marinesco-Sjögren syndrome (MSS), as being robustly expressed in disease-resistant slow motor neurons but not in ER stress-prone fast-fatigable motor neurons. In a mouse model of MSS, we demonstrate impaired ER homeostasis in motor neurons in response to loss of SIL1 function. Loss of a single functional Sil1 allele in an ALS mouse model (SOD1-G93A) enhanced ER stress and exacerbated ALS pathology. In SOD1-G93A mice, SIL1 levels were progressively and selectively reduced in vulnerable fast-fatigable motor neurons. Mechanistically, reduction in SIL1 levels was associated with lowered excitability of fast-fatigable motor neurons, further influencing expression of specific ER chaperones. Adeno-associated virus-mediated delivery of SIL1 to familial ALS motor neurons restored ER homeostasis, delayed muscle denervation and prolonged survival.