RESUMEN
The gene and cell therapy field saw its first approved treatments in Europe in 2012 and the United States in 2017 and is projected to be at least a $10B USD industry by 2025. Despite this success, a massive gap exists between the companies, clinics, and researchers developing these therapeutic approaches, and their availability to the patients who need them. The unacceptable reality is a geographic exclusion of low-and middle-income countries (LMIC) in gene therapy development and ultimately the provision of gene therapies to patients in LMIC. This is particularly relevant for gene therapies to treat human immunodeficiency virus infection and hemoglobinopathies, global health crises impacting tens of millions of people primarily located in LMIC. Bridging this divide will require research, clinical and regulatory infrastructural development, capacity-building, training, an approval pathway and community adoption for success and sustainable affordability. In 2020, the Global Gene Therapy Initiative was formed to tackle the barriers to LMIC inclusion in gene therapy development. This working group includes diverse stakeholders from all sectors and has set a goal of introducing two gene therapy Phase I clinical trials in two LMIC, Uganda and India, by 2024. Here we report on progress to date for this initiative.
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Países en Desarrollo , Infecciones por VIH , Humanos , Estados UnidosRESUMEN
AIMS/HYPOTHESIS: To overcome the donor shortage in the treatment of advanced type 1 diabetes by islet transplantation, human embryonic stem cells (hESCs) show great potential as an unlimited alternative source of beta cells. hESCs may have immune privileged properties and it is important to determine whether these properties are preserved in hESC-derived cells. METHODS: We comprehensively investigated interactions of both innate and adaptive auto- and allo-immunity with hESC-derived pancreatic progenitor cells and hESC-derived endocrine cells, retrieved after in-vivo differentiation in capsules in the subcutis of mice. RESULTS: We found that hESC-derived pancreatic endodermal cells expressed relatively low levels of HLA endorsing protection from specific immune responses. HLA was upregulated when exposed to IFNγ, making these endocrine progenitor cells vulnerable to cytotoxic T cells and alloreactive antibodies. In vivo-differentiated endocrine cells were protected from complement, but expressed more HLA and were targets for alloreactive antibody-dependent cellular cytotoxicity and alloreactive cytotoxic T cells. After HLA compatibility was provided by transduction with HLA-A2, preproinsulin-specific T cells killed insulin-producing cells. CONCLUSIONS/INTERPRETATION: hESC-derived pancreatic progenitors are hypoimmunogenic, while in vivo-differentiated endocrine cells represent mature targets for adaptive immune responses. Our data support the need for immune intervention in transplantation of hESC-derived pancreatic progenitors. Cell-impermeable macro-encapsulation may suffice.
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Células Madre Embrionarias Humanas/inmunología , Células Secretoras de Insulina/inmunología , Células Madre/metabolismo , Inmunidad Adaptativa/inmunología , Aloinjertos , Autoinmunidad , Células Cultivadas , Antígeno HLA-A2 , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inmunidad Humoral/inmunología , Inmunidad Innata/inmunología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Interferón gamma/metabolismoRESUMEN
These preliminary data from an ongoing first-in-human phase 1/2, open-label study provide proof-of-concept that pluripotent stem cell-derived pancreatic endoderm cells (PEC-01) engrafted in type 1 diabetes patients become islet cells releasing insulin in a physiologically regulated fashion. In this study of 17 subjects aged 22-57 with type 1 diabetes, PEC-01 cells were implanted subcutaneously in VC-02 macroencapsulation devices, allowing for direct vascularization of the cells. Engraftment and insulin expression were observed in 63% of VC-02 units explanted from subjects at 3-12 months post-implant. Six of 17 subjects (35.3%) demonstrated positive C-peptide as early as 6 months post-implant. Most reported adverse events were related to surgical implant or explant procedures (27.9%) or to side-effects of immunosuppression (33.7%). Initial data suggest that pluripotent stem cells, which can be propagated to the desired biomass and differentiated into pancreatic islet-like tissue, may offer a scalable, renewable alternative to pancreatic islet transplants.
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Péptido C/metabolismo , Células Inmovilizadas/citología , Diabetes Mellitus Tipo 1/terapia , Endodermo/citología , Insulina/metabolismo , Páncreas/citología , Trasplante de Células Madre , Células Madre/citología , Adolescente , Adulto , Anciano , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.
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Acetilcolina/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Inhibición Neural/fisiología , Unión Neuromuscular/fisiología , Agregación de Receptores/fisiología , Receptores Colinérgicos/fisiología , Agrina/deficiencia , Agrina/farmacología , Animales , Western Blotting/métodos , Bungarotoxinas/farmacocinética , Carbacol/farmacología , Carbocianinas/farmacocinética , Línea Celular , Colina O-Acetiltransferasa/deficiencia , Agonistas Colinérgicos/farmacología , Quinasa 5 Dependiente de la Ciclina , Diafragma/citología , Interacciones Farmacológicas , Embrión de Mamíferos , Femenino , Proteínas de Homeodominio , Inmunohistoquímica/métodos , Inmunoprecipitación , Hibridación in Situ/métodos , Ratones , Ratones Noqueados , Muscarina/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Liso/efectos de los fármacos , Músculo Liso/embriología , Inhibición Neural/efectos de los fármacos , Embarazo , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Agregación de Receptores/efectos de los fármacos , Roscovitina , Sinaptofisina/metabolismo , Factores de Tiempo , Factores de Transcripción/deficienciaRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) or its naturally occurring analog, neurturin (NTN), can potentially improve the function and delay the rate of degeneration of dopaminergic neurons in Parkinson's disease (PD). However, their delivery to the central nervous system has proven to be a significant challenge. Viral vector-mediated gene transfer offers a practical means to continuously supply neurotrophic factors in targeted areas of the brain. CERE-120 is an adeno-associated viral vector encoding NTN, developed for the treatment of PD. We found that the kinetics and pattern of NTN expression in the rat striatum following injection of CERE-120 is rapid, increases significantly up to 4 weeks, and exhibits a stable volume of distribution thereafter for at least 1 year, the longest time-point evaluated. Quantitative enzyme-linked immunosorbent assay confirmed that steady-state levels are maintained from 4 weeks onward. We demonstrated that NTN volume of distribution can be controlled by varying the dose of vector injected and that NTN delivered via CERE-120 was bioactive, as evidenced by the neuroprotection of DA neurons in the rat 6-hydroxydopamine lesion model. These data provided the foundation for further non-clinical development of CERE-120, leading to an ongoing clinical trial in PD patients.
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Dependovirus/genética , Dopamina/metabolismo , Vectores Genéticos/genética , Degeneración Nerviosa/genética , Neurturina/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/terapia , Animales , Línea Celular , Modelos Animales de Enfermedad , Dopamina/análogos & derivados , Expresión Génica , Terapia Genética , Genoma Viral/genética , Humanos , Cinética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Degeneración Nerviosa/terapia , Neurturina/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Type 1 diabetes (T1D) is characterized by destruction of glucose-responsive insulin-producing pancreatic ß-cells and exhibits immune infiltration of pancreatic islets, where CD8 lymphocytes are most prominent. Curative transplantation of pancreatic islets is seriously hampered by the persistence of autoreactive immune cells that require high doses of immunosuppressive drugs. An elegant approach to confer graft protection while obviating the need for immunosuppression is the use of encapsulation devices that allow for the transfer of oxygen and nutrients, yet prevent immune cells from making direct contact with the islet grafts. Here we demonstrate that macroencapsulation devices (TheraCyte) loaded with neonatal pancreatic tissue and transplanted into RIP-LCMV.GP mice prevented disease onset in a model of virus-induced diabetes mellitus. Histological analyses revealed that insulin-producing cells survived within the device in animal models of diabetes. Our results demonstrate that these encapsulation devices can protect from an immune-mediated attack and can contain a sufficient amount of insulin-producing cells to prevent overt hyperglycemia.
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Diabetes Mellitus Tipo 1/terapia , Hiperglucemia/prevención & control , Trasplante de Islotes Pancreáticos/inmunología , Trasplante de Islotes Pancreáticos/métodos , Animales , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/virología , Modelos Animales de Enfermedad , Hiperglucemia/inmunología , Hiperglucemia/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine (ACh), is thought to be present in kinetic excess in cholinergic neurons. The rate-limiting factor in ACh production is the provision of choline to ChAT. Cholinergic neurons are relatively unique in their expression of the choline transporter 1 (CHT1), which exhibits high-affinity for choline and catalyzes its uptake from the extracellular space to the neuron. Multiple lines of evidence indicate that the activity of CHT1 is a key determinant of choline supply for ACh synthesis. We examined the interaction of ChAT and ChT activity using mice heterozygous for a null mutation in the Chat gene (Chat+/-). In these mice, brain ChAT activity was reduced by 40-50% relative to the wild type, but brain ACh levels as well as ACh content and depolarization-evoked ACh release in hippocampal slices were normal. However, the amount of choline taken up by CHT1 and ACh synthesized de novo from choline transported by CHT1 in hippocampal slices, as well as levels of CHT1 mRNA in the septum and CHT1 protein in several regions of the CNS, were 50-100% higher in Chat+/- than in Chat+/+ mice. Thus, haploinsufficiency of ChAT leads to an increased expression of CHT1. Increased ChT activity may compensate for the reduced ChAT activity in Chat+/- mice, contributing to the maintenance of apparently normal cholinergic function as reflected by normal performance of these mice in several behavioral assays.
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Encéfalo/metabolismo , Colina O-Acetiltransferasa/genética , Proteínas de Transporte de Membrana/biosíntesis , Acetilcolina/metabolismo , Animales , Conducta Animal , Transporte Biológico , Colina O-Acetiltransferasa/biosíntesis , Colina O-Acetiltransferasa/deficiencia , Hipocampo/metabolismo , Técnicas In Vitro , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Mutantes , ARN Mensajero/biosíntesis , Tabique del Cerebro/metabolismo , Regulación hacia ArribaRESUMEN
In this study we examined the developmental roles of acetylcholine (ACh) by establishing and analyzing mice lacking choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh. As predicted, ChAT-deficient embryos lack both spontaneous and nerve-evoked postsynaptic potentials in muscle and die at birth. In mutant embryos, abnormally increased nerve branching occurs on contact with muscle, and hyperinnervation continues throughout subsequent prenatal development. Postsynaptically, ACh receptor clusters are markedly increased in number and occupy a broader muscle territory in the mutants. Concomitantly, the mutants have significantly more motor neurons than normal. At an ultrastructural level, nerve terminals are smaller in mutant neuromuscular junctions, and they make fewer synaptic contacts to the postsynaptic muscle membrane, although all of the typical synaptic components are present in the mutant. These results indicate that ChAT is uniquely essential for the patterning and formation of mammalian neuromuscular synapses.
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Colina O-Acetiltransferasa/deficiencia , Enfermedades Neuromusculares/patología , Unión Neuromuscular/patología , Animales , Recuento de Células , Supervivencia Celular , Colina O-Acetiltransferasa/genética , Diafragma/embriología , Diafragma/inervación , Diafragma/patología , Potenciales Postsinápticos Excitadores/genética , Marcación de Gen , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neuronas Motoras/patología , Enfermedades Neuromusculares/congénito , Enfermedades Neuromusculares/genética , Unión Neuromuscular/ultraestructura , ARN Mensajero/biosíntesis , Agregación de Receptores , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Transmisión Sináptica/genética , Sinaptofisina/biosíntesisRESUMEN
Type 1 diabetes (T1D) is an autoimmune disease caused by T cell-mediated destruction of insulin-producing ß cells in the islets of Langerhans. In most cases, reversal of disease would require strategies combining islet cell replacement with immunotherapy that are currently available only for the most severely affected patients. Here, we demonstrate that immunotherapies that target T cell costimulatory pathways block the rejection of xenogeneic human embryonic-stem-cell-derived pancreatic endoderm (hESC-PE) in mice. The therapy allowed for long-term development of hESC-PE into islet-like structures capable of producing human insulin and maintaining normoglycemia. Moreover, short-term costimulation blockade led to robust immune tolerance that could be transferred independently of regulatory T cells. Importantly, costimulation blockade prevented the rejection of allogeneic hESC-PE by human PBMCs in a humanized model in vivo. These results support the clinical development of hESC-derived therapy, combined with tolerogenic treatments, as a sustainable alternative strategy for patients with T1D.
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Diabetes Mellitus Tipo 1/terapia , Endodermo/citología , Endodermo/inmunología , Células Madre Embrionarias Humanas/trasplante , Tolerancia Inmunológica/inmunología , Páncreas/citología , Linfocitos T Reguladores/inmunología , Animales , Diabetes Mellitus Tipo 1/patología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Páncreas/inmunología , Trasplante HeterólogoRESUMEN
Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.
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Técnicas de Cultivo Celular por Lotes/métodos , Diferenciación Celular/fisiología , Diabetes Mellitus Tipo 1/terapia , Células Madre Embrionarias/citología , Células Madre Embrionarias/trasplante , Células Secretoras de Insulina/citología , Análisis de Varianza , Animales , Criopreservación/métodos , Células Madre Embrionarias/fisiología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones SCID , EstreptozocinaRESUMEN
Huntington's disease (HD) is a devastating neurodegenerative disease characterized by the selective loss of neurons in the striatum and cerebral cortex. This study tested the hypothesis that an adenoassociated viral (AAV2) vector encoding for the trophic factor neurturin (NTN) could provide neuroprotection in the rat 3-nitropropionic acid (3NP) model of HD. Rats received AAV2-NTN (CERE-120), AAV2-eGFP or Vehicle, followed 4 weeks later by the mitochondrial toxin 3NP. 3NP induced motor impairments were observed on the rotarod test, the platform test, and a clinical rating scale in all groups. However, each of these deficits was attenuated by AAV2-NTN (CERE-120). Stereological counts revealed a significant protection of NeuN-ir striatal neurons from 3NP toxicity by AAV2-NTN. These data support the concept that AAV2-NTN might be a valuable treatment for patients with Huntington's disease.
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Cuerpo Estriado/metabolismo , Terapia Genética/métodos , Enfermedad de Huntington/terapia , Degeneración Nerviosa/terapia , Neuronas/metabolismo , Neurturina/genética , Animales , Recuento de Células , Muerte Celular/genética , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/fisiopatología , Citoprotección/genética , Proteínas de Unión al ADN , Dependovirus/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen/tendencias , Vectores Genéticos/genética , Enfermedad de Huntington/inducido químicamente , Enfermedad de Huntington/genética , Masculino , Actividad Motora/genética , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/prevención & control , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Neurotoxinas , Neurturina/uso terapéutico , Nitrocompuestos , Proteínas Nucleares/metabolismo , Propionatos , Ratas , Ratas Endogámicas Lew , Recuperación de la Función/genética , Resultado del TratamientoRESUMEN
Neurturin (NTN) is a neurotrophic factor with known potential to protect and restore the function of dopaminergic substantia nigra neurons whose degeneration has been most closely linked to the major motor deficits in Parkinson's disease (PD). CERE-120, an adeno-associated virus serotype 2 (AAV2)-based gene delivery vector encoding human NTN, is being developed as a potential therapeutic for PD. In a series of preclinical studies reported herein, CERE-120 delivery to the striatum produced a dose-related neuroprotection of nigrostriatal neurons in the rat 6-hydroxydopamine (6-OHDA) lesion model. Long-lasting efficacy of CERE-120 was evidenced by substantia nigra cell protection, preserved fiber innervation of the striatum, and behavioral recovery for at least 6 months. In addition, striatal infusion of CERE-120 was found to have a safety and tolerability profile devoid of side effects or toxicological responses, for at least 12 months post-treatment, even at dose multiples 125 times that of the lowest efficacious dose tested. These results support the ongoing CERE-120 clinical program in PD patients.
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Adenoviridae/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Neurturina/genética , Trastornos Parkinsonianos/terapia , Animales , Conducta Animal , Cuerpo Estriado/citología , Cuerpo Estriado/fisiología , Modelos Animales de Enfermedad , Dopamina/fisiología , Femenino , Vectores Genéticos/toxicidad , Humanos , Masculino , Fibras Nerviosas/fisiología , Fármacos Neuroprotectores , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/fisiopatología , Ratas , Ratas Sprague-Dawley , Sustancia Negra/citología , Sustancia Negra/fisiología , SimpaticolíticosRESUMEN
Huntington's disease (HD) is a fatal, genetic, neurological disorder resulting from a trinucleotide repeat expansion in the gene that encodes for the protein huntingtin. These excessive repeats confer a toxic gain of function on huntingtin, which leads to the degeneration of striatal and cortical neurons and a devastating motor, cognitive, and psychological disorder. Trophic factor administration has emerged as a compelling potential therapy for a variety of neurodegenerative disorders, including HD. We previously demonstrated that viral delivery of glial cell line-derived neurotrophic factor (GDNF) provides structural and functional neuroprotection in a rat neurotoxin model of HD. In this report we demonstrate that viral delivery of GDNF into the striatum of presymptomatic mice ameliorates behavioral deficits on the accelerating rotorod and hind limb clasping tests in transgenic HD mice. Behavioral neuroprotection was associated with anatomical preservation of the number and size of striatal neurons from cell death and cell atrophy. Additionally, GDNF-treated mice had a lower percentage of neurons containing mutant huntingtin-stained inclusion bodies, a hallmark of HD pathology. These data further support the concept that viral vector delivery of GDNF may be a viable treatment for patients suffering from HD.
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Técnicas de Transferencia de Gen , Factores Neurotróficos Derivados de la Línea Celular Glial/metabolismo , Enfermedad de Huntington/patología , Enfermedad de Huntington/fisiopatología , Fármacos Neuroprotectores/metabolismo , Animales , Conducta Animal/fisiología , Muerte Celular , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Femenino , Terapia Genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factores Neurotróficos Derivados de la Línea Celular Glial/genética , Factores Neurotróficos Derivados de la Línea Celular Glial/uso terapéutico , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/terapia , Cuerpos de Inclusión/química , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Proteínas Nucleares/metabolismo , RatasRESUMEN
The Cre/loxP system is increasingly showing promise for investigating genes involved in neural function. Here, we demonstrate that in vivo modification of genes in the mouse brain can be accomplished in a spatial- and temporal-specific manner by targeted delivery of an adeno-associated virus (AAV) encoding a green fluorescent protein/Cre recombinase (GFP/Cre) fusion protein. By using a reporter mouse, in which Cre recombinase activates beta-galactosidase expression, we demonstrate long-term recombination of neurons in the hippocampus, striatum, and septum as early as 7 days after stereotaxic injection of virus. Recombined cells were observed for at least 6 months postinjection without evidence of cell loss or neural damage. AAV-mediated delivery of GFP/Cre provides a valuable approach to alter the mouse genome, as AAV delivers genes efficiently to neurons with low toxicity. This approach will greatly facilitate the study of genetic modifications in the mouse brain.