RESUMEN

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that exceed the AAV capacity.

Asunto(s)

Distrofina , Terapia Genética , Inteínas , Distrofia Muscular de Duchenne , Empalme de Proteína , Animales , Humanos , Masculino , Ratones , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Distrofina/biosíntesis , Distrofina/deficiencia , Distrofina/genética , Distrofina/metabolismo , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Inteínas/genética , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Empalme de Proteína/genética

RESUMEN

Limb-girdle muscular dystrophy type R9 (LGMDR9) is a muscle-wasting disease that begins in the hip and shoulder regions of the body. This disease is caused by mutations in fukutin-related protein (FKRP), a glycosyltransferase critical for maintaining muscle cell integrity. Here we investigated potential gene therapies for LGMDR9 containing an FKRP expression construct with untranslated region (UTR) modifications. Initial studies treated an aged dystrophic mouse model (FKRPP448L) with adeno-associated virus vector serotype 6 (AAV6). Grip strength improved in a dose- and time-dependent manner, injected mice exhibited fewer central nuclei and serum creatine kinase levels were 3- and 5-fold lower compared to those in non-injected FKRPP448L mice. Treatment also partially stabilized the respiratory pattern during exercise and improved treadmill running, partially protecting muscle from exercise-induced damage. Western blotting of C2C12 myotubes using a novel rabbit antibody confirmed heightened translation with the UTR modifications. We further explored the question of FKRP toxicity in wild-type mice using high doses of two additional muscle-tropic capsids: AAV9 and AAVMYO1. No toxic effects were detected with either therapeutic agent. These data further support the feasibility of gene therapy to treat LGMDR9.

RESUMEN

Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young animals with limited muscle fibrosis and wasting, thereby favoring muscle transduction, myonuclear editing, and prevention of disease progression. Here, we explore muscle-specific dystrophin gene editing following intramuscular delivery of AAV6:CK8e-CRISPR/SaCas9 in 3- and 8-year-old dystrophic CXMD dogs and provide a qualitative comparison to AAV6:CK8e-micro-dystrophin gene replacement at 6 weeks post-treatment. Gene editing restored the dystrophin reading frame in ∼1.3% of genomes and in up to 4.0% of dystrophin transcripts following excision of a 105-kb mutation containing region spanning exons 6-8. However, resulting dystrophin expression levels and effects on muscle pathology were greater with the use of micro-dystrophin gene transfer. This study demonstrates that our muscle-specific multi-exon deletion strategy can correct a frequently mutated region of the dystrophin gene in an aged large animal DMD model, but underscores that further enhancements are required to reach efficiencies comparable to AAV micro-dystrophin. Our observations also indicate that treatment efficacy and state of muscle pathology at the time of intervention are linked, suggesting the need for additional methodological optimizations related to age and disease progression to achieve relevant clinical translation of CRISPR-based therapies to all DMD patients.

Asunto(s)

Distrofina , Distrofia Muscular de Duchenne , Envejecimiento , Animales , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Perros , Distrofina/genética , Edición Génica/métodos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia

RESUMEN

Vectors derived from adeno-associated viruses (AAV) have been generated using numerous naturally occurring and synthetic serotypes of the virus. Such vectors have proven to be extremely useful for a variety of gene transfer studies, both in vitro and in vivo, and are increasingly being used in gene therapy protocols for a variety of human disorders. Methods to produce AAV vectors typically rely on co-transfection of several different plasmid vectors that carry the transgene of interest (the gene to be delivered , in a "transfer plasmid") and helper genes needed for AAV vector replication and packaging (helper plasmids). While the methods used to generate AAV are conceptually simple, minor variations in a variety of steps can result in significant differences in the overall yield of vector. Here we describe protocols for generating vectors derived from AAV6, which are particularly useful for gene transfer to muscle tissues.

Asunto(s)

Dependovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Animales , Replicación del ADN/genética , Vectores Genéticos/genética , Humanos , Músculos/metabolismo , Músculos/patología , Transgenes/genética

RESUMEN

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.

Asunto(s)

Distrofina/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Animales , Western Blotting , Línea Celular , Perros , Distrofina/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Microscopía Electrónica , Microscopía Fluorescente , Distrofia Muscular de Duchenne/genética

RESUMEN

JSRV, a simple beta-retrovirus, is the etiologic agent of ovine pulmonary adenocarcinoma, a form of non-small cell lung cancer in sheep and goats. It has been shown that the envelope protein alone is sufficient to induce tumorigenesis in the lungs of mice when delivered via an adeno-associated viral vector. Here, we tested the hypothesis that JSRV envelope-induced tumors are maintained by a small population of tumor-initiating cells, termed cancer stem cells. To test this hypothesis, dissociated cancer cells were sorted from envelope-induced tumors in mouse lung based on the putative stem cell markers Sca-1, CD34, and CD133, the pluripotency-associated transcription factor Oct4, and the level of Wnt signaling. No association with increased tumor-initiating capacity was found with any of the cell-surface markers. In addition, we were unable to detect any evidence of Oct4 expression in tumor-bearing mouse lung. However, tumor cells possessing an active Wnt signaling pathway did show a significant correlation with increased tumor formation upon transplantation. Limiting dilution transplant analysis suggests the existence of a large fraction of cells with the ability to propagate tumor growth, with increasing tumor initiation potential correlating with activated Wnt signaling.

Asunto(s)

Adenocarcinoma/inducido químicamente , Productos del Gen env , Retrovirus Ovino Jaagsiekte/química , Neoplasias Pulmonares/inducido químicamente , Células Madre Neoplásicas/fisiología , Vía de Señalización Wnt/fisiología , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Células Cultivadas , Progresión de la Enfermedad , Retrovirus Ovino Jaagsiekte/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Células 3T3 NIH , Células Madre Neoplásicas/patología , Ovinos , Enfermedades de las Ovejas/inducido químicamente , Enfermedades de las Ovejas/genética , Enfermedades de las Ovejas/patología , Activación Transcripcional/fisiología , Vía de Señalización Wnt/genética

RESUMEN

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats, while the closely related enzootic nasal tumor virus type 1 (ENTV-1) and ENTV-2 induce tumors in the nasal epithelium of sheep and goats, respectively. When expressed using a strong Rous sarcoma virus promoter, the envelope proteins of these viruses induce tumors in the respiratory tract of mice, but only in the distal airway. To examine the role of the retroviral long terminal repeat (LTR) promoters in determining tissue tropism, adeno-associated virus (AAV) vectors expressing alkaline phosphatase under the control of the JSRV, ENTV-1, or ENTV-2 LTRs were generated and administered to mice. The JSRV LTR was active in all airway epithelial cells, while the ENTV LTRs were active in the nasal epithelium and alveolar type II cells but poorly active in tracheal and bronchial epithelial cells. When vectors were administered systemically, the ENTV-1 and -2 LTRs were inactive in major organs examined, whereas the JSRV showed high-level activity in the liver. When a putative transcriptional enhancer from the 3' end of the env gene was inserted upstream of the JSRV and ENTV-1 LTRs in the AAV vectors, a dramatic increase in transgene expression was observed. However, intranasal administration of AAV vectors containing any combination of ENTV or JSRV LTRs and Env proteins induced tumors only in the lower airway. Our results indicate that mice do not provide an adequate model for nasal tumor induction by ENTV despite our ability to express genes in the nasal epithelium.

Asunto(s)

Bronquios/virología , Regulación Viral de la Expresión Génica/fisiología , Retrovirus Ovino Jaagsiekte/fisiología , Neoplasias Pulmonares/virología , Regiones Promotoras Genéticas , Alveolos Pulmonares/patología , Tráquea/virología , Infecciones Tumorales por Virus/genética , Animales , Secuencia de Bases , Bronquios/microbiología , Cartilla de ADN , Dependovirus/genética , Vectores Genéticos , Neoplasias Pulmonares/patología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tráquea/microbiología

RESUMEN

We evaluated the potential of lung-directed gene therapy for alpha1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans.

Asunto(s)

Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Pulmón/metabolismo , alfa 1-Antitripsina/genética , Animales , Líquido del Lavado Bronquioalveolar , ADN Complementario , Perros , Ensayo de Inmunoadsorción Enzimática , Terapia Genética , Humanos , Ratones , Deficiencia de alfa 1-Antitripsina/terapia

RESUMEN

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues has been shown to depend heavily on the AAV type from which the vector capsid proteins are derived. Among the AAV types studied, AAV6 efficiently transduces cells of the airway epithelium, making it a good candidate for the treatment of lung diseases such as cystic fibrosis. Here we have evaluated the effects of various promoter sequences on transduction rates and gene expression levels in the lung. Of the strong viral promoters examined, the Rous sarcoma virus (RSV) promoter performed significantly better than a human cytomegalovirus (CMV) promoter in the airway epithelium. However, a hybrid promoter consisting of a CMV enhancer, beta-actin promoter and splice donor, and a beta-globin splice acceptor (CAG promoter) exhibited even higher expression than either of the strong viral promoters alone, showing a 38-fold increase in protein expression over the RSV promoter. In addition, we show that vectors containing either the RSV or CAG promoter expressed well in the nasal and tracheal epithelium. Transduction rates in the 90% range were achieved in many airways with the CAG promoter, showing that with the proper AAV capsid proteins and promoter sequences, efficient transduction can be achieved.

Asunto(s)

Fosfatasa Alcalina/genética , Dependovirus/genética , Vectores Genéticos/genética , Regiones Promotoras Genéticas/genética , Transducción Genética , Fosfatasa Alcalina/análisis , Empalme Alternativo , Animales , Citomegalovirus/genética , Globinas/genética , Humanos , Pulmón/enzimología , Pulmón/metabolismo , Ratones , Distribución Tisular

RESUMEN

Jaagsiekte sheep retrovirus (JSRV) induces bronchioalveolar tumors in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse airway epithelial cells induces similar tumors, indicating that Env expression is sufficient for tissue-specific tumor formation. Enzootic nasal tumor virus (ENTV) is related to JSRV but induces tumors in the nasal epithelium of sheep and goats. Here we found that ENTV Env can also induce tumors in mice but, unexpectedly, with a phenotype identical to that of tumors induced by the JSRV Env, indicating that factors other than Env mediate the tissue specificity of tumor induction by ENTV.

Asunto(s)

Neoplasias de los Bronquios/virología , Productos del Gen env/química , Retrovirus Ovino Jaagsiekte/metabolismo , Pulmón/virología , Animales , Bronquios/virología , ADN Viral/genética , Dependovirus/genética , Vectores Genéticos , Ratones , Ratones Noqueados , Modelos Genéticos , Regiones Promotoras Genéticas , Virus/metabolismo

RESUMEN

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy directed to the lungs, in particular for treatment of cystic fibrosis (CF). In animal models of lung gene transfer, neutralizing antibodies in serum made in response to vector exposure have been associated with a partial to complete block to repeat transduction by vectors with the same capsid type, thus transduction by AAV vectors might be inefficient in humans previously exposed to the same AAV type. AAV type 2 (AAV2) has been used in clinical trials of lung gene transfer, but AAV5 and AAV6 have been shown to mediate more efficient transduction in rodent lungs and in cultured human airway epithelia compared with that of AAV2. Here we have measured neutralizing antibodies against AAV type 2, 5, and 6 vectors in serum from children and adults with CF, and from normal adults. About 30% of adults were seropositive for AAV2, 20-30% were seropositive for AAV6, and 10-20% were seropositive for AAV5. CF children were seropositive for AAV type 2, 5, or 6 at rates of 4-15%. All individuals seropositive for AAV6 were also seropositive for AAV2, and the AAV6 titer was low compared with the AAV2 titer. AAV5-positive sera were lower both in titers and rates than those seen for AAV6. The results indicate that AAV type 2, 5 or 6 exposure is low in CF and control populations and even lower in CF children.

Asunto(s)

Anticuerpos Antivirales/sangre , Fibrosis Quística/inmunología , Dependovirus/inmunología , Terapia Genética , Vectores Genéticos , Adolescente , Adulto , Animales , Anticuerpos Antivirales/inmunología , Niño , Preescolar , Fibrosis Quística/sangre , Fibrosis Quística/terapia , Fibrosis Quística/virología , Dependovirus/genética , Genoma Viral , Humanos , Lactante , Recién Nacido , Pulmón/metabolismo , Ratones , Estudios Seroepidemiológicos

RESUMEN

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues is determined primarily by the viral capsid proteins. In contrast to vectors made with AAV type 2 capsids, those having type 5 or 6 capsids show high transduction rates in airway epithelial cells, in a range that should be sufficient for treating lung disease. Here we have compared the properties of vectors made with AAV5 or AAV6 capsid proteins to determine whether their receptor usage is similar, and found several differences between the viruses. First, an AAV6 vector did not hemagglutinate red blood cells, whereas an AAV5 vector did, and this property was sialic acid dependent. Second, AAV5 vector transduction required sialic acid in all cells tested, whereas AAV6 vector transduction was sialic acid dependent or independent, depending on the target cells tested. Third, levels of an AAV6 vector that interfered with entry of another AAV6 vector only poorly inhibited AAV5 vector transduction and vice versa. These results indicate that AAV5 and AAV6 vectors use distinct cellular receptors for cell entry. Although both AAV5 and AAV6 vectors exhibited high transduction rates in well-differentiated human airway epithelial cultures, they exhibited distinct cell-type transduction profiles in mouse lung that may reflect differences in receptor usage.

Asunto(s)

Proteínas de la Cápside/fisiología , Dependovirus/fisiología , Técnicas de Transferencia de Gen , Receptores Virales/fisiología , Transducción Genética , Animales , Células CHO , Línea Celular Transformada , Línea Celular Tumoral , Cricetinae , Dependovirus/inmunología , Dependovirus/patogenicidad , Células Epiteliales/virología , Expresión Génica , Terapia Genética , Hemaglutinación por Virus , Humanos , Riñón/citología , Riñón/embriología , Pulmón/citología , Macaca mulatta , Ratones , Mucosa Nasal/citología , Neuraminidasa/metabolismo

RESUMEN

BACKGROUND: Adeno-associated virus (AAV) vectors are attractive tools for direct intralumenal arterial gene transfer in interventional cardiology or cardiovascular surgery, but clinical application has been constrained by poor gene expression in this setting. METHODS: To improve arterial wall gene expression, a hybrid promoter consisting of a cytomegalovirus (CMV) immediate-early enhancer, a chicken beta-actin transcription start site, and a rabbit beta-globin intron (CAG promoter) was substituted for the Rous sarcoma virus (RSV) promoter in an AAV type 2 vector with an alkaline phosphatase (AP) reporter gene. RESULTS: Intralumenal transduction of rabbit carotid arteries by an AAV2 vector containing a CAG promoter resulted in gene expression in a mean of > or = 80% of the lumenal area at 14 days following exposure, compared to < or = 25% gene-expressing area with the RSV promoter-based control vector. The high prevalence of gene expression was maintained at 3, 7, 14, and 28 days. Importantly, in carotid arteries transduced with the CAG promoter, gene product expression was readily visible by the third day following transduction whereas gene expression was rarely seen before day 10 using the RSV promoter in the same animal model. On histology, AP gene expression was predominantly in vascular smooth muscle cells although some endothelial cell expression was also present. CONCLUSIONS: Substituting the CAG for the RSV promoter results in widespread gene expression, demonstrating efficient arterial wall transduction by AAV2 vectors. This finding plus the early time to gene expression hold promise for AAV vectors as agents for direct intralumenal arterial wall gene delivery during cardiovascular interventions.

Asunto(s)

Actinas/genética , Arterias Carótidas/metabolismo , Citomegalovirus/genética , Dependovirus/genética , Globinas/genética , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Células Cultivadas , Pollos , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Técnicas de Transferencia de Gen , Genes Reporteros , Vectores Genéticos , Humanos , Técnicas In Vitro , Plásmidos , Regiones Promotoras Genéticas , Conejos

RESUMEN

Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats, with significant animal health and economic consequences. The host range of JSRV is in part limited by species-specific differences in the virus entry receptor, hyaluronidase 2 (Hyal2), which is not functional as a receptor in mice but is functional in humans. Sheep are immunotolerant of JSRV because of the expression of closely related endogenous retroviruses, which are not present in humans and most other species, and this may facilitate oncogenesis. Here we show that expression of the JSRV envelope (Env) protein alone in lungs of mice, by using a replication-incompetent adeno-associated virus vector, results in tumours with a bronchiolo-alveolar localization like those seen in sheep. Whereas lethal disease was observed in immunodeficient mice, tumour development was almost entirely blocked in immunocompetent mice. Our results provide a rare example of an oncogenic viral structural protein, show that interaction of the viral Env protein with the virus entry receptor Hyal2 is not required for tumorigenesis, and indicate that immune recognition of Env can protect against JSRV tumorigenesis.

Asunto(s)

Productos del Gen env/metabolismo , Retrovirus Ovino Jaagsiekte/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/virología , Enfermedades de las Ovejas/patología , Enfermedades de las Ovejas/virología , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Productos del Gen env/genética , Productos del Gen env/inmunología , Genoma Viral , Humanos , Retrovirus Ovino Jaagsiekte/genética , Retrovirus Ovino Jaagsiekte/inmunología , Pulmón/patología , Pulmón/virología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/veterinaria , Ratones , Receptores Virales/metabolismo , Ovinos/virología , Especificidad de la Especie

RESUMEN

Vectors based on recombinant adeno-associated viruses (rAAV) have emerged as tools of choice for gene transfer to skeletal muscle. rAAV vectors demonstrate efficient, safe, and stable transduction. Multiple serotypes of AAV exist, but vectors based on serotype 2 (rAAV2) are the most thoroughly characterized and frequently employed. Here, we characterize transduction of the skeletal musculature using rAAV vectors pseudotyped with serotype 6 capsid proteins (rAAV6). We demonstrate that rAAV6 vectors can efficiently transduce the skeletal musculature of mice at levels >500-fold higher than is achievable with rAAV2 vectors and can readily saturate individual muscles following direct injection. Further, rAAV6 vectors are capable of transducing the diaphragm and intercostal muscles of mice after a simple injection into the intrathoracic cavity and are capable of widespread transduction throughout the musculature of mice injected in the intraperitoneal space as newborn pups. These results demonstrate that rAAV6 vectors hold great potential for use in gene delivery protocols targeting the skeletal musculature.

Asunto(s)

Dependovirus/genética , Vectores Genéticos , Músculo Esquelético/metabolismo , Transducción Genética , Animales , Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros/genética , Vectores Genéticos/administración & dosificación , Inyecciones Intraperitoneales , Ratones , Fibras Musculares Esqueléticas/química , beta-Galactosidasa/análisis , beta-Galactosidasa/genética

RESUMEN

Jaagsiekte sheep retrovirus (JSRV) infects lung epithelial cells in sheep, and oncoretroviral vectors bearing JSRV Env can mediate transduction of human cells, suggesting that such vectors might be useful for lung-directed gene therapy. Here we show that JSRV Env can also efficiently pseudotype a human immunodeficiency virus type 1-based lentiviral vector, a more suitable vector for transduction of slowly dividing lung epithelial cells. We created several chimeric Env proteins that, unlike the parental Env, do not transform rodent fibroblasts but are still capable of pseudotyping lentiviral and oncoretroviral vectors.

Asunto(s)

Productos del Gen env/metabolismo , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/fisiología , VIH-1/genética , VIH-1/fisiología , Retrovirus Ovino Jaagsiekte/metabolismo , Secuencia de Aminoácidos , Animales , Moléculas de Adhesión Celular/metabolismo , División Celular , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/virología , Fibroblastos , Proteínas Ligadas a GPI , Productos del Gen env/química , Productos del Gen env/genética , Humanos , Hialuronoglucosaminidasa/metabolismo , Retrovirus Ovino Jaagsiekte/genética , Pulmón/citología , Pulmón/virología , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/genética , Virus de la Leucemia Murina de Moloney/fisiología , Especificidad de Órganos , Plásmidos/genética , Receptores Virales/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Roedores , Transducción Genética

RESUMEN

The ability of adeno-associated viral (AAV) vectors to promote persistent gene expression in nondividing cells in multiple somatic tissues of animals (1-4) makes them excellent tools for gene transfer. One tissue of interest for gene transfer is the lung epithelium, which is afflicted in cystic fibrosis (CF). However, although initial animal studies done with vectors based on AAV type 2 have demonstrated transduction in multiple cells types in the lung, the rates were modest in alveolar cells and much lower rates in airway epitheila and required high particle numbers (5-7). In contrast, an AAV6 encapsidated vector showed preferential transduction of epithelial cells in large and small airways (8) at rates that exceeded the 5% efficiency rate predicted to have a therapeutic value for CF gene therapy (9). In fact, recent studies using vectors based on other AAV types showed that types 1-6 have different tissue tropisms (10-15), and that types 5 and 6 are more efficient than type 2 in lung epithelium (8,14). In mouse lung, an AAV2 vector gave modest transduction rates.

Asunto(s)

Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Pulmón/metabolismo , Animales , Ratones , Transducción Genética

RESUMEN

The small packaging capacity of adeno-associated virus (AAV) vectors limits the utility of this promising vector system for transfer of large genes. We explored the possibility that larger genes could be reconstituted following homologous recombination between AAV vectors carrying overlapping gene fragments. An alkaline phosphatase (AP) gene was split between two such AAV vectors (rec vectors) and packaged using AAV2 or AAV6 capsid proteins. Rec vectors having either capsid protein recombined to express AP in cultured cells at about 1-2% of the rate observed for an intact vector. Surprisingly, the AAV6 rec vectors transduced lung cells in mice almost as efficiently as did an intact vector, with 10% of airway epithelial cells, the target for treatment of cystic fibrosis (CF), being positive. Thus AAV rec vectors may be useful for diseases such as CF that require transfer of large genes.

Asunto(s)

Dependovirus/genética , Vectores Genéticos/genética , Recombinación Genética , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , Transducción Genética/métodos , Animales , Proteínas de la Cápside/genética , Regulación de la Expresión Génica , Humanos , Pulmón/citología , Pulmón/fisiología , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Mucosa Respiratoria/virología