RESUMO
Osteoarthritis results in chronic pain and loss of function. Proinflammatory cytokines create both osteoarthritis pathology and pain. Current treatments are poorly effective, have significant side effects, and have not targeted the cytokines central to osteoarthritis development and maintenance. Interleukin-10 is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. However, interleukin-10 protein has a short half-life in vivo and poor joint permeability. For sustained IL-10 activity, we developed a plasmid DNA-based therapy that expresses a long-acting human interleukin-10 variant (hIL-10var). Here, we describe the 6-month GLP toxicology study of this therapy. Intra-articular injections of hIL-10var pDNA into canine stifle joints up to 1.5 mg bilaterally were well-tolerated and without pathologic findings. This represents the first long-term toxicologic assessment of intra-articular pDNA therapy. We also report results of a small double-blind, placebo-controlled study of the effect of intra-articular hIL-10var pDNA on pain measures in companion (pet) dogs with naturally occurring osteoarthritis. This human IL-10-based targeted therapy reduced pain measures in the dogs, based on veterinary and owner ratings, without any adverse findings. These results with hIL-10var pDNA therapy, well-tolerated and without toxicologic effects, establish the basis for clinical trials of a new class of safe and effective therapies for OA.
Assuntos
Osteoartrite do Joelho , Osteoartrite , Animais , Cães , Terapia Genética , Interleucina-10 , Osteoartrite/terapia , Dor , PlasmídeosRESUMO
Relapsing-remitting multiple sclerosis is commonly associated with motor impairments, neuropathic pain, fatigue, mood disorders, and decreased life expectancy. However, preclinical pharmacological studies predominantly rely on clinical scoring of motor deficit as the sole behavioral endpoint. Thus, the translational potential of these studies is limited. Here, we have assessed the therapeutic potential of a novel anti-inflammatory interleukin-10 (IL-10) non-viral gene therapy formulation (XT-101-R) in a rat relapsing remitting experimental autoimmune encephalomyelitis (EAE) model. EAE induced motor deficits and neuropathic pain as reflected by induction of low-threshold mechanical allodynia, suppressed voluntary wheel running, decreased social exploration, and was associated with markedly enhanced mortality. We also noted that voluntary wheel running was depressed prior to the onset of motor deficit, and may therefore serve as a predictor of clinical symptoms onset. XT-101-R was intrathecally dosed only once at the onset of motor deficits, and attenuated each of the EAE-induced symptoms and improved survival, relative to vehicle control. This is the first pharmacological assessment of such a broad range of EAE symptoms, and provides support for IL-10 gene therapy as a clinical strategy for the treatment of multiple sclerosis.
Assuntos
Ansiedade/psicologia , Ansiedade/terapia , Comportamento Animal/efeitos dos fármacos , Encefalomielite Autoimune Experimental/psicologia , Encefalomielite Autoimune Experimental/terapia , Fadiga/psicologia , Fadiga/terapia , Interleucina-10/genética , Neuralgia/psicologia , Neuralgia/terapia , Animais , Comportamento Exploratório , Terapia Genética , Hiperalgesia/psicologia , Hiperalgesia/terapia , Injeções Espinhais , Relações Interpessoais , Expectativa de Vida , Masculino , Atividade Motora , RatosRESUMO
Alcohol use disorder (AUD) exacts enormous personal, social and economic costs globally. Return to alcohol use in treatment-seeking patients with AUD is common, engendered by a cycle of repeated abstinence-relapse episodes even with use of currently available pharmacotherapies. Repeated ethanol use induces dopaminergic signaling neuroadaptations in ventral tegmental area (VTA) neurons of the mesolimbic reward pathway, and sustained dysfunction of reward circuitry is associated with return to drinking behavior. We tested this hypothesis by infusing adeno-associated virus serotype 2 vector encoding human glial-derived neurotrophic factor (AAV2-hGDNF), a growth factor that enhances dopaminergic neuron function, into the VTA of four male rhesus monkeys, with another four receiving vehicle, following induction of chronic alcohol drinking. GDNF expression ablated the return to alcohol drinking behavior over a 12-month period of repeated abstinence-alcohol reintroduction challenges. This behavioral change was accompanied by neurophysiological modulations to dopamine signaling in the nucleus accumbens that countered the hypodopaminergic signaling state associated with chronic alcohol use, indicative of a therapeutic modulation of limbic circuits countering the effects of alcohol. These preclinical findings suggest gene therapy targeting relapse prevention may be a potential therapeutic strategy for AUD.
Assuntos
Alcoolismo , Animais , Masculino , Consumo de Bebidas Alcoólicas/genética , Consumo de Bebidas Alcoólicas/metabolismo , Alcoolismo/terapia , Alcoolismo/tratamento farmacológico , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Etanol/metabolismo , Etanol/farmacologia , Etanol/uso terapêutico , Terapia Genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Núcleo Accumbens/metabolismo , Primatas/genética , Área Tegmentar Ventral/metabolismoRESUMO
Clinical trials involving direct infusion of neurotrophic therapies for Parkinson's disease (PD) have suffered from poor coverage of the putamen. The planned use of a novel interventional-magnetic resonance imaging (iMRI) targeting system for achieving precise, real-time convection-enhanced delivery in a planned clinical trial of adeno-associated virus serotype 2 (AAV2)-glial-derived neurotrophic factor (GDNF) in PD patients was modeled in nonhuman primates (NHP). NHP received bilateral coinfusions of gadoteridol (Gd)/AAV2-GDNF into two sites in each putamen, and three NHP received larger infusion volumes in the thalamus. The average targeting error for cannula tip placement in the putamen was <1 mm, and adjacent putamenal infusions were distributed in a uniform manner. GDNF expression patterns in the putamen were highly correlated with areas of Gd distribution seen on MRI. The distribution volume to infusion volume ratio in the putamen was similar to that in the thalamus, where larger infusions were achieved. Modeling the placement of adjacent 150 and 300 µl thalamic infusions into the three-dimensional space of the human putamen demonstrated coverage of the postcommissural putamen, containment within the striatum and expected anterograde transport to globus pallidus and substantia nigra pars reticulata. The results elucidate the necessary parameters for achieving widespread GDNF expression in the putamenal motor area and afferent substantia nigra of PD patients.
Assuntos
Dependovirus/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Doença de Parkinson/terapia , Putamen/metabolismo , Animais , Ensaios Clínicos como Assunto , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Macaca mulatta , Imageamento por Ressonância Magnética , Doença de Parkinson/patologiaRESUMO
We are creating families of designer G protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (G(s), G(i) and G(q)). We review these advances here to facilitate the use of these powerful and diverse tools.
Assuntos
Engenharia de Proteínas/métodos , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Evolução Molecular , Humanos , Ligantes , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genéticaRESUMO
Despite many decades of drug development, effective therapies for neuropathic pain remain elusive. The recent recognition of spinal cord glia and glial pro-inflammatory cytokines as important contributors to neuropathic pain suggests an alternative therapeutic strategy; that is, targeting glial activation or its downstream consequences. While several glial-selective drugs have been successful in controlling neuropathic pain in animal models, none are optimal for human use. Thus the aim of the present studies was to explore a novel approach for controlling neuropathic pain. Here, an adeno-associated viral (serotype II; AAV2) vector was created that encodes the anti-inflammatory cytokine, interleukin-10 (IL-10). This anti-inflammatory cytokine is known to suppress the production of pro-inflammatory cytokines. Upon intrathecal administration, this novel AAV2-IL-10 vector was successful in transiently preventing and reversing neuropathic pain. Intrathecal administration of an AAV2 vector encoding beta-galactosidase revealed that AAV2 preferentially infects meningeal cells surrounding the CSF space. Taken together, these data provide initial support that intrathecal gene therapy to drive the production of IL-10 may prove to be an efficacious treatment for neuropathic pain.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Mediadores da Inflamação/fisiologia , Interleucina-10/biossíntese , Interleucina-10/genética , Nervo Isquiático/fisiopatologia , Ciática/prevenção & controle , Ciática/fisiopatologia , Animais , Dependovirus/fisiologia , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Vetores Genéticos/uso terapêutico , Humanos , Inflamação/metabolismo , Inflamação/prevenção & controle , Inflamação/virologia , Injeções Espinhais , Interleucina-10/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Ciática/metabolismoRESUMO
Propentofylline is a phosphodiesterase inhibitor that has been shown to attenuate the onset of morphine tolerance when administered intrathecally to rats. The present studies examined whether systemic administration could be effective in attenuating morphine tolerance in non-injured rodents using a similar dosing paradigm. Propentofylline at 10, 30, or 50 mg/kg, administered intraperitoneally once daily for 5 days, was unable to attenuate morphine tolerance established by twice daily administration of 10 mg/kg morphine. These results suggest that direct delivery of propentofylline to the central nervous system (CNS) may be required in order to attenuate morphine tolerance.
Assuntos
Tolerância a Medicamentos , Morfina/administração & dosagem , Dor/tratamento farmacológico , Xantinas/administração & dosagem , Analgesia/métodos , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Injeções Intraperitoneais , Injeções Espinhais , Masculino , Fármacos Neuroprotetores/administração & dosagem , Dor/prevenção & controle , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Ferimentos e LesõesRESUMO
BACKGROUND: Liposomal drug packaging is well established as an effective means for increasing drug half-life, sustaining drug activity, and increasing drug efficacy, whether administered locally or distally to the site of disease. However, information regarding the relative effectiveness of peripheral (distal) versus local administration of liposomal therapeutics is limited. This issue is of importance with respect to the treatment of central nervous system cancer, for which the blood-brain barrier presents a significant challenge in achieving sufficient drug concentration in tumors to provide treatment benefit for patients. METHODS: We compared the anti-tumor activity and efficacy of a nanoliposomal formulation of irinotecan when delivered peripherally by vascular route with intratumoral administration by convection-enhanced delivery (CED) for treating intracranial glioblastoma xenografts in athymic mice. RESULTS: Our results show significantly greater anti-tumor activity and survival benefit from CED of nanoliposomal irinotecan. In 2 of 3 efficacy experiments, there were animal subjects that experienced apparent cure of tumor from local administration of therapy, as indicated by a lack of detectable intracranial tumor through bioluminescence imaging and histopathologic analysis. Results from investigating the effectiveness of combination therapy with nanoliposomal irinotecan plus radiation revealed that CED administration of irinotecan plus radiation conferred greater survival benefit than did irinotecan or radiation monotherapy and also when compared with radiation plus vascularly administered irinotecan. CONCLUSIONS: Our results indicate that liposomal formulation plus direct intratumoral administration of therapeutic are important for maximizing the anti-tumor effects of irinotecan and support clinical trial evaluation of this therapeutic plus route of administration combination.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Camptotecina/análogos & derivados , Sistemas de Liberação de Medicamentos , Glioblastoma/tratamento farmacológico , Lipossomos , Nanopartículas , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Camptotecina/administração & dosagem , Convecção , Vias de Administração de Medicamentos , Feminino , Glioblastoma/mortalidade , Glioblastoma/patologia , História Antiga , Humanos , Técnicas Imunoenzimáticas , Injeções Intraperitoneais , Irinotecano , Camundongos , Camundongos Nus , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Convection-enhanced delivery (CED) of substances within the human brain is becoming a more frequent experimental treatment option in the management of brain tumors, and more recently in phase 1 trials for gene therapy in Parkinson's disease (PD). Benefits of this intracranial drug-transfer technology include a more efficient delivery of large volumes of therapeutic agent to the target region when compared with more standard delivery approaches (i.e., biopolymers, local infusion). In this article, we describe specific technical modifications we have made to the CED process to make it more effective. For example, we developed a reflux-resistant infusion cannula that allows increased infusion rates to be used. We also describe our efforts to visualize the CED process in vivo, using liposomal nanotechnology and real-time intraoperative MRI. In addition to carrying the MRI contrast agent, nanoliposomes also provide a standardized delivery vehicle for the convection of drugs to a specific brain-tissue volume. This technology provides an added level of assurance via visual confirmation of CED, allowing intraoperative alterations to the infusion if there is reflux or aberrant delivery. We propose that these specific modifications to the CED technology will improve efficacy by documenting and standardizing the treatment-volume delivery. Furthermore, we believe that this image-guided CED platform can be used in other translational neuroscience efforts, with eventual clinical application beyond neuro-oncology and PD.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Imageamento por Ressonância Magnética/métodos , Doenças do Sistema Nervoso/terapia , HumanosRESUMO
The main medication for idiopathic Parkinson disease is L-Dopa. Drug efficacy declines steadily in part because the converting enzyme, aromatic L-amino acid decarboxylase (AADC), is lost concomitant with substantia nigra atrophy. Over the past decade, we have developed a gene therapy approach in which AADC activity is restored to the brain by infusion into the striatum of a recombinant adeno-associated virus carrying human AADC cDNA. We report here the results of an investigation of the relationship between vector dose and a series of efficacy markers, such as PET, L-Dopa response, and AADC enzymatic activity. At low doses of vector, no effect of vector was seen on PET or behavioral response. At higher doses, a sharp improvement in both parameters was observed, resulting in an approximate 50% improvement in L-Dopa responsiveness. The relationship between vector dose and AADC enzymatic activity in tissue extracts was linear. We conclude that little behavioral improvement can be seen until AADC activity reaches a level that is no longer rate limiting for conversion of clinical doses of L-Dopa into dopamine or for trapping of the PET tracer FMT. These findings have implications for the design and interpretation of clinical studies of AAV-hAADC gene therapy.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Dependovirus/genética , Terapia Genética , Doença de Parkinson/genética , Doença de Parkinson/terapia , Animais , Descarboxilases de Aminoácido-L-Aromático/genética , Comportamento Animal , Humanos , Macaca mulatta , Doença de Parkinson/enzimologia , Tomografia por Emissão de Pósitrons , Fatores de TempoRESUMO
Striatal neurons convert L-dopa to dopamine (DA) following gene transfer of aromatic L-amino acid decarboxylase (AADC) via adeno-associated virus (AAV) in parkinsonian monkeys. We investigated whether AAV-AADC could reduce or eliminate L-dopa-induced dyskinesias (LIDs) and side effects in MPTP-treated monkeys. Five monkeys were made parkinsonian by bilateral MPTP lesions. The optimal therapeutic dose of L-dopa was determined using an acute dose response regimen. After 3 weeks of chronic L-dopa treatment, AAV-AADC or control vector was bilaterally injected into the striatum. Animals were assessed for 6 months with the same L-dopa dosing as presurgery as well as chronic oral L-dopa treatment. Presurgery LID was observed at doses greater than 5 mg/kg. The AAV-AADC-treated animals displayed an average 7.3-fold decrease in the therapeutic dose of L-dopa throughout the 6-month follow-up period. Only AAV-AADC-treated monkeys were susceptible to dyskinesias even at sub-clinical doses. Immunohistochemical analysis revealed well-delineated foci of AADC within the striatum. These results suggest that high levels of focal DA were generated in response to L-dopa administration and may be responsible for the exacerbation of dyskinesias. This may be similar to focal dopaminergic activity in PD patients that developed off-drug or "runaway" dyskinesias following fetal mesencephalic grafts.
Assuntos
Antiparkinsonianos/efeitos adversos , Dopamina/efeitos adversos , Levodopa/efeitos adversos , Neurônios/metabolismo , Doença de Parkinson Secundária/induzido quimicamente , Animais , Antiparkinsonianos/administração & dosagem , Descarboxilases de Aminoácido-L-Aromático/fisiologia , Comportamento Animal/fisiologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Dependovirus/fisiologia , Modelos Animais de Doenças , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Seguimentos , Técnicas de Transferência de Genes , Imuno-Histoquímica/métodos , Levodopa/administração & dosagem , Intoxicação por MPTP/complicações , Intoxicação por MPTP/tratamento farmacológico , Macaca mulatta , Imageamento por Ressonância Magnética/métodos , Doença de Parkinson Secundária/patologia , Tomografia por Emissão de Pósitrons/métodos , Fatores de TempoRESUMO
Recombinant AAV vectors containing a dimerizer-inducible system of transcriptional activation provide a strategy for control of therapeutic gene expression in the CNS. Here we explored this system for regulated expression of human aromatic L-amino acid decarboxylase (hAADC) in a rodent model of Parkinson disease. Expression of hAADC, the enzyme that converts L-dopa to dopamine, was dependent on reconstitution of a functional transcription factor (TF) by the dimerizer rapamycin. Two vectors, AAV-CMV-TF and AAV-Z12-hAADC, were infused into striata of 6-OHDA-lesioned rats. Rapamycin-induced increases in expression of hAADC repeatedly produced robust rotational behavior in response to low doses of L-dopa. Seven weeks after vector infusion, AADC expression in brain was quantitated by both stereology and Western blot analysis following the final rapamycin treatment. While a low level of hAADC was observed in rats that were not induced with rapamycin, this basal expression was not significant enough to elicit a rotational response to L-dopa. This study demonstrated a robust behavioral response of parkinsonian rats to regulated hAADC expression. Recombinant AAV vectors controlled by rapamycin or its analogs show promise as candidates for CNS therapies in which regulation of the transgene is desired.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/biossíntese , Dependovirus/genética , Oxidopamina , Doença de Parkinson Secundária/terapia , Comportamento Estereotipado , Animais , Antiparkinsonianos/farmacologia , Linhagem Celular , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Dimerização , Modelos Animais de Doenças , Dopamina/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica , Terapia Genética , Vetores Genéticos , Humanos , Levodopa/farmacologia , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/metabolismo , Ratos , Ratos Sprague-Dawley , Recombinação Genética , Sirolimo/farmacologia , Comportamento Estereotipado/efeitos dos fármacos , Transdução GenéticaRESUMO
Although L-DOPA is the drug of choice for Parkinson's disease, prolonged L-DOPA therapy results in decreased drug effectiveness and the appearance of motor complications. This may be due in part to the progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC). We have developed an adeno-associated virus vector (AAV-hAADC) that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats and monkeys improves L-DOPA responsiveness by improving AADC-mediated conversion of L-DOPA to dopamine. This is now the basis of a proposed therapy for advanced Parkinson's disease. A key concern has been that over-production of dopamine in striatal neurons could cause dopamine toxicity. To investigate this possibility in a controlled system, mixed striatal primary rat neuronal cultures were prepared. Exposure of cultures to high concentrations of L-DOPA induced the following changes: cell death in nigral and striatal neurons, aggregation of neurofilaments and focal axonal swellings, abnormal expression of DARPP-32, and activation of astroglia and microglial cells. Transduction of cultures with AAV-hAADC resulted in efficient and sustained neuronal expression of the AADC protein and prevented all the L-DOPA-induced toxicities. The protective effects were due primarily to AADC-dependent conversion of L-DOPA to dopamine and an increase in induction of vesicular monoamine transporter resulting in dopamine storage in cultured cells. These results suggest a neuroprotective role for AADC gene transfer against L-DOPA toxicity.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/genética , Corpo Estriado/enzimologia , Dependovirus/genética , Técnicas de Transferência de Genes , Levodopa/toxicidade , Animais , Descarboxilases de Aminoácido-L-Aromático/administração & dosagem , Linhagem Celular , Células Cultivadas , Corpo Estriado/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Citoproteção/fisiologia , Vetores Genéticos , Humanos , Ratos , Ratos Sprague-DawleyRESUMO
Gene therapy for the control of pain has, to date, targeted neurons. However, recent evidence supports that spinal cord glia are critical to the creation and maintenance of pain facilitation through the release of proinflammatory cytokines. Because of the ability of interleukin-10 (IL-10) to suppress proinflammatory cytokines, we tested whether an adenoviral vector encoding human IL-10 (AD-h-IL10) would block and reverse pain facilitation. Three pain models were examined, all of which are mediated by spinal pro-inflammatory cytokines. Acute intrathecal administration of rat IL-10 protein itself briefly reversed chronic constriction injury-induced mechanical allodynia and thermal hyperalgesia. The transient reversal caused by IL-10 protein paralleled the half-life of human IL-10 protein in the intrathecal space (t(1/2) approximately 2 h). IL-10 gene therapy both prevented and reversed thermal hyperalgesia and mechanical allodynia, without affecting basal responses to thermal or mechanical stimuli. Extra-territorial, as well as territorial, pain changes were reversed by this treatment. Intrathecal AD-h-IL10 injected over lumbosacral spinal cord led to elevated lumbosacral cerebrospinal fluid (CSF) levels of human IL-10, with far less human IL-10 observed in cervical CSF. In keeping with IL-10's known anti-inflammatory actions, AD-h-IL10 lowered CSF levels of IL-1, relative to control AD. These studies support that this gene therapy approach provides an alternative to neuronally focused drug and gene therapies for clinical pain control.
Assuntos
Terapia Genética/métodos , Interleucina-10/uso terapêutico , Manejo da Dor , Adenoviridae/genética , Animais , Comportamento Animal , Relação Dose-Resposta a Droga , Lateralidade Funcional/fisiologia , Vetores Genéticos , Membro Posterior/efeitos dos fármacos , Membro Posterior/inervação , Membro Posterior/fisiopatologia , Humanos , Injeções Espinhais/métodos , Interleucina-1/biossíntese , Interleucina-1/uso terapêutico , Interleucina-10/biossíntese , Interleucina-10/líquido cefalorraquidiano , Interleucina-10/genética , Masculino , Microinjeções/métodos , Dor/classificação , Dor/etiologia , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-1/administração & dosagem , Receptores Tipo I de Interleucina-1 , Fatores de Tempo , Zimosan/uso terapêuticoRESUMO
In this study, a modified infusion procedure and a novel infusion device designed for use in humans (Clinical Device B) were evaluated for delivery of recombinant adeno-associated virus (AAV2) to brain. The device is composed of 1.2 m of fused silica inserted through a 24.6-cm surgical steel cannula designed to fit a standard Leksell clinical stereotaxic frame and micro-infusion syringe pump. AAV2 encoding the human aromatic l-amino acid decarboxylase gene (AAV-hAADC-2) was infused into the putamen of 4 normal rhesus monkeys as a supportive study for a clinical trial in Parkinson's disease (PD) patients. Two infusion protocols were tested: a ramped procedure (slow stepwise increases in rate from 0.2 muL/min to 1 muL/min), thought to be essential for convection-enhanced delivery (CED), and a non-ramped infusion at a constant rate of 1 muL/min. The primary endpoints were safety evaluation of the infusion procedures and assessment of transgene expression at 5.5 weeks post-infusion. Clinical observations after vector infusions revealed no behavioral abnormalities during the study period. No differences in gross pathology with either the ramped or non-ramped infusion procedure were observed. Histopathology of the putamen was comparable with both procedures, and revealed only minimal localized inflammatory tissue reaction along the needle track in response to cannula placement and vector infusion. AADC immunohistochemistry demonstrated that vector was distributed throughout the putamen, with no significant difference in volume of immunostaining with either infusion procedure. Serum antibody levels against AAV2 vector exhibited a minor increase after infusion. These results validate the clinical utility of this new infusion device and non-ramped infusion conditions for intraputamenal gene therapy, and have the potential to impact a number of human diseases in which delivery of therapeutics to brain is indicated.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Macaca mulatta/cirurgia , Doença de Parkinson/terapia , Putamen/cirurgia , Transfecção/métodos , Animais , Descarboxilases de Aminoácido-L-Aromático/genética , Encefalite/etiologia , Encefalite/patologia , Encefalite/fisiopatologia , Desenho de Equipamento , Regulação da Expressão Gênica/genética , Terapia Genética/efeitos adversos , Terapia Genética/instrumentação , Vetores Genéticos/genética , Bombas de Infusão/efeitos adversos , Macaca mulatta/anatomia & histologia , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Putamen/patologia , Putamen/fisiopatologia , Recuperação de Função Fisiológica/genética , Seringas/efeitos adversos , Seringas/normas , Transgenes/genética , Resultado do TratamentoRESUMO
We tested the hypotheses that initial immunization of rats with rAAV might limit subsequent transduction by rAAV-hAADC when stereotaxically infused into the striatum and that the level of inhibition would correlate with AAV neutralizing antibody titers. Immunohistochemical detection of AADC and analysis by stereology revealed that the control group (no immunization) had the greatest volume of distribution of AADC (20.32 +/- 2.03 mm3) (+/-SD). There was a 58% decrease in spread (8.46 +/- 3.67 mm3, P < 0.008) in the high-dose immunization group (5 x 10(10) vg rAAV-null). Transduction weakly correlated with preexisting titer levels of neutralizing antibody at the time of intrastriatal rAAV-hAADC infusion. Only rats with neutralizing antibody titers of 1:1208 +/- 332 had significantly decreased AADC transgene expression compared to the unimmunized control group. Immunohistochemistry on serial sections for inflammatory markers including GFAP, CD11b, CD4, and CD8a revealed normal morphology and no cellular infiltration, suggesting little immune reaction in the CNS. We conclude that rAAV vectors can transduce brain tissue in the context of preexisting immunity, but that efficiency of transduction declines significantly in the presence of very high titers of neutralizing antibodies. These results have important implications for gene therapy for CNS disorders.