RESUMEN
Galectin-3 (Gal-3), a ß-galactoside-binding lectin, serves as a pattern-recognition receptor (PRR) of dendritic cells (DCs) in regulating proinflammatory cytokine production. Galectin-3 (Gal-3) siRNA downregulates expression of IL-6, IL-1ß and IL-23 p19, while upregulates IL-10 and IL-12 p35 in TLR/NLR stimulated human MoDCs. Furthermore, Gal-3 siRNA-treated MoDCs enhanced IFN-γ production in SEB-stimulated CD45RO CD4 T-cells, but attenuated IL-17A and IL-5 production by CD4 T-cells. Addition of neutralizing antibodies against Gal-3, or recombinant Gal-3 did not differentially modulate IL-23 p19 versus IL-12 p35. The data indicate that intracellular Gal-3 acts as cytokine hub of human DCs in responding to innate immunity signals. Gal-3 downregulation reprograms proinflammatory cytokine production by MoDCs that inhibit Th2/Th17 development.
Asunto(s)
Citocinas/biosíntesis , Células Dendríticas/inmunología , Galectina 3/biosíntesis , Inflamación/inmunología , Receptores de Reconocimiento de Patrones/genética , Diferenciación Celular/inmunología , Línea Celular , Células Dendríticas/citología , Regulación hacia Abajo , Galectina 3/genética , Células HT29 , Humanos , Interleucina-10/biosíntesis , Subunidad p35 de la Interleucina-12/biosíntesis , Interleucina-1beta/biosíntesis , Subunidad p19 de la Interleucina-23/biosíntesis , Interleucina-6/biosíntesis , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Reconocimiento de Patrones/biosíntesis , Células Th17/citología , Células Th17/inmunología , Células Th2/citología , Células Th2/inmunologíaRESUMEN
Mutations in the Conserved Oligomeric Golgi (COG) complex give rise to type II congenital disorders of glycosylation (CDG). Thus far, mutations have been identified in 6 of the 8 COG subunits. Here we present data identifying a previously reported CDG-IIx case from Singapore as a new COG4 patient with 2 novel mutations leading to p.E233X and p.L773R; with p.E233X being a de novo mutation. As a result, COG4 protein expression was dramatically reduced, while expression of the other subunits remained unaffected. Analysis of serum N-glycans revealed deficiencies in both sialylation and galactosylation. Furthermore, patient fibroblasts have impaired O-glycosylation. Importantly, patient fibroblasts exhibited a delay in Brefeldin A (BFA) induced retrograde transport, a common characteristic seen in COG deficiencies.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/metabolismo , Brefeldino A/farmacología , Trastornos Congénitos de Glicosilación/fisiopatología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Prueba de Complementación Genética , Aparato de Golgi/metabolismo , Aparato de Golgi/patología , Humanos , Lentivirus/genética , Mutación , Polisacáridos/análisis , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas/efectos de los fármacos , SingapurRESUMEN
BACKGROUND: At present, it is highly controversial whether pure mast cells can serve as antigen presenting cells, and it is not known whether the capacity of antigen presenting function is temporally restricted to a particular subset of differentiated mast cells. Evidence is presented for a novel surface FcepsilonRIhi , MHC II +, and c-kit + pure mast cell subset, temporally restricted as antigen-presenting cells in the immune axis of T-cell activation. RESULTS: Bone marrow-derived mast cells (BMMC) cultured in the presence of IL-3 for three weeks are pure mast cells based on surface expression of lineage-specific marker, c-kit and FcepsilonRI. Herein we present the first demonstration that approximately 98.7% c-kit + and FcepsilonRI expressing BMMC, further depleted of any contaminated professional antigen-presenting cells, are still fully capable of presenting antigens, i.e., OVA protein, OVA peptide, and IgE-TNP-OVA, to OVA peptide-specific T-cell hybridomas. Notably, IgE-dependent antigen presentation is more efficient compared to that resulting from direct antigen uptake. Importantly, we present the novel finding that only surface FcepsilonRIhi mast cells, also expressing surface MHC II exhibited antigen-presenting function. In contrast, surface FcepsilonRIlo mast cells without expressing surface MHC II were not capable of antigen presentation. Interestingly, the antigen-presenting function of BMMC was irrevocably lost during the third and fourth week in IL-3 or SCF containing cultures. CONCLUSIONS: This is the first observation to attribute a spatiotemporally restricted antigen-presenting function to a subset of three-week old pure BMMC expressing both high levels of surface FcepsilonRI and surface MHC II. We propose that mast cells play an important role in immune deviating and/or sustaining the activation of infiltrating CD4 T-cells, and modulating T-cell mediated allergic inflammation via its flexibility to present antigens and antigen-IgE complexes.
Asunto(s)
Presentación de Antígeno/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Mastocitos/inmunología , Receptores de IgE/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos/inmunología , Células Cultivadas , Femenino , Espacio Intracelular/metabolismo , Mastocitos/citología , Ratones , Factores de TiempoRESUMEN
CONTEXT: Inborn errors in protein glycosylation, such as the congenital disorders of glycosylation (CDGs), generate multifaceted syndromes that impair many organ systems. We here report the diagnosis of the third known patient with CDG-Id. RESULTS: The patient's phenotype was extremely severe, and she succumbed at 19 d of age. Leading features included hyperinsulinemic hypoglycemia, and autopsy revealed islet cell hyperplasia with increased beta-cell mass. Other features were a Dandy-Walker malformation, facial dysmorphisms, and profound hypotonia. The patient carried a novel homozygous point mutation (512G>A) in the hALG3 gene, which encodes a mannosyltransferase. Lentiviral complementation with wild-type hALG3 corrects the biochemical defect in the patient's fibroblasts. CONCLUSIONS: Our findings underscore the importance of proper glycosylation in several major organ systems and emphasize that CDG should be ruled out in patients with persistent hyperinsulinemic hypoglycemia of unknown etiology.
Asunto(s)
Hiperinsulinismo/genética , Hipoglucemia/genética , Islotes Pancreáticos/patología , Manosiltransferasas/genética , Errores Innatos del Metabolismo/genética , Adulto , Células Cultivadas , Femenino , Glicosilación , Humanos , Hiperplasia , Inmunohistoquímica , Recién Nacido , EmbarazoRESUMEN
BACKGROUND: Congenital disorders of glycosylation (CDGs) are inherited diseases caused by glycosylation defects. Incorrectly glycosylated proteins induce protein misfolding and endoplasmic reticulum (ER) stress. The most common form of CDG, PMM2-CDG, is caused by deficiency in the cytosolic enzyme phosphomannomutase 2 (PMM2). Patients with PMM2-CDG exhibit a significantly reduced number of cerebellar Purkinje cells and granule cells. The molecular mechanism underlying the specific cerebellar neurodegeneration in PMM2-CDG, however, remains elusive. RESULTS: Herein, we report that cerebellar granule cells (CGCs) are more sensitive to tunicamycin (TM)-induced inhibition of total N-glycan synthesis than cortical neurons (CNs). When glycan synthesis was inhibited to a comparable degree, CGCs exhibited more cell death than CNs. Furthermore, downregulation of PMM2 caused more CGCs to die than CNs. Importantly, we found that upon PMM2 downregulation or TM treatment, ER-stress response proteins were elevated less significantly in CGCs than in CNs, with the GRP78/BiP level showing the most significant difference. We further demonstrate that overexpression of GRP78/BiP rescues the death of CGCs resulting from either TM-treatment or PMM2 downregulation. CONCLUSIONS: Our results indicate that the selective susceptibility of cerebellar neurons to N-glycosylation defects is due to these neurons' inefficient response to ER stress, providing important insight into the mechanisms of selective neurodegeneration observed in CDG patients.
Asunto(s)
Cerebelo/patología , Trastornos Congénitos de Glicosilación/patología , Estrés del Retículo Endoplásmico , Degeneración Nerviosa/patología , Neuronas/patología , Animales , Muerte Celular/efectos de los fármacos , Corteza Cerebral/patología , Trastornos Congénitos de Glicosilación/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glicosilación/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfotransferasas (Fosfomutasas)/metabolismo , Tunicamicina/toxicidadRESUMEN
Senile plaques consisting of beta-amyloid (Abeta) and neurofibrillary tangles composed of hyperphosphorylated tau are major pathological hallmarks of Alzheimer's disease (AD). Elucidation of factors that modulate Abeta generation and tau hyperphosphorylation is crucial for AD intervention. Here, we identify a mouse gene Rps23r1 that originated through retroposition of ribosomal protein S23. We demonstrate that RPS23R1 protein reduces the levels of Abeta and tau phosphorylation by interacting with adenylate cyclases to activate cAMP/PKA and thus inhibit GSK-3 activity. The function of Rps23r1 is demonstrated in cells of various species including human, and in transgenic mice overexpressing RPS23R1. Furthermore, the AD-like pathologies of triple transgenic AD mice were improved and levels of synaptic maker proteins increased after crossing them with Rps23r1 transgenic mice. Our studies reveal a new target/pathway for regulating AD pathologies and uncover a retrogene and its role in regulating protein kinase pathways.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas tau/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Secuencia de Bases , Encéfalo/metabolismo , Línea Celular Tumoral , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Células PC12 , Fosforilación , Ratas , Proteínas Ribosómicas/genética , Homología de Secuencia de Ácido Nucleico , Sinapsis/metabolismoRESUMEN
Treatment of degenerating basal forebrain cholinergic neurons with nerve growth factor (NGF) in Alzheimer's disease has long been contemplated, but an effective and safe delivery method has been lacking. Towards achieving this goal, we are currently developing CERE-110, an adeno-associated virus-based gene delivery vector that encodes for human NGF, for stereotactic surgical delivery to the human nucleus basalis of Meynert. Results indicate that NGF transgene delivery to the targeted brain region via CERE-110 is reliable and accurate, that NGF transgene distribution can be controlled by altering CERE-110 dose, and that it is possible to achieve restricted NGF expression limited to but covering the target brain region. Results from animals examined at longer time periods of 3, 6, 9 and 12 months after CERE-110 delivery indicate that NGF transgene expression is stable and sustained at all time points, with no loss or build-up of protein over the long-term. In addition, results from a series of experiments indicate that CERE-110 is neuroprotective and neurorestorative to basal forebrain cholinergic neurons in the rat fimbria-fornix lesion and aged rat models, and has bioactive effects on young rat basal forebrain cholinergic neurons. These findings, as well as those from several additional non-clinical experiments conducted in both rats and monkeys, led to the initiation of a Phase I clinical study to evaluate the safety and efficacy of CERE-110 in Alzheimer's disease subjects, which is currently ongoing.
Asunto(s)
Fibras Colinérgicas/metabolismo , Fibras Colinérgicas/virología , Sistemas de Liberación de Medicamentos/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/metabolismo , Factor de Crecimiento Nervioso/administración & dosificación , Prosencéfalo/metabolismo , Prosencéfalo/virología , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Humanos , Masculino , Marmota , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/genética , Degeneración Nerviosa/prevención & control , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/uso terapéutico , Prosencéfalo/citología , Ratas , Ratas Endogámicas F344 , Ratas Sprague-DawleyRESUMEN
We present two sibs with congenital disorder of glycosylation (CDG) type Id. Each shows severe global delay, failure to thrive, seizures, microcephaly, axial hypotonia, and disaccharidase deficiency. One sib has more severe digestive issues, while the other is more neurologically impaired. Each is compound heterozygous for a novel point mutation and an already known mutation in the ALG3 gene that leads to the synthesis of a severely truncated oligosaccharide precursor for N-glycans. The defect is corrected by introduction of a normal ALG3 cDNA. CDG should be ruled out in all patients with severe seizures and failure to thrive. (c) 2007 Wiley-Liss, Inc.
Asunto(s)
Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/genética , Manosiltransferasas/genética , Hermanos , Ceguera/diagnóstico , Ceguera/etiología , Niño , Trastornos Congénitos de Glicosilación/complicaciones , Análisis Mutacional de ADN , ADN Complementario/genética , Diagnóstico Diferencial , Femenino , Prueba de Complementación Genética , Glicosilación , Heterocigoto , Humanos , Masculino , Hipotonía Muscular/diagnóstico , Hipotonía Muscular/etiología , Mutación , Fenotipo , Convulsiones/diagnóstico , Convulsiones/etiologíaRESUMEN
In this report, we describe a brother and sister who presented at birth with short-limb skeletal dysplasia, polyhydramnios, prematurity, and generalized edema. Dysmorphic features included broad nose, thick ears, thin lips, micrognathia, inverted nipples, ulnar deviation at the wrists, spatulate fingers, fifth finger camptodactyly, nail hypoplasia, and talipes equinovarus. Other features included short stature, microcephaly, psychomotor retardation, B-cell lymphopenic hypogammaglobulinemia, sensorineural deafness, retinal detachment and blindness, intestinal malrotation with poor gastrointestinal motility, persistent hyponatremia, intermittent hypoglycemia, and thrombocytopenia. Cardiac anomalies included PDA, VSD, hypertrophic cardiomyopathy, and arrhythmias. The brother had a small penis with hypospadias, hypoplastic scrotum, and non-palpable testes. Skeletal findings included absent ossification of cervical vertebral bodies, pubic bones, knee epiphyses, and tali. Both sibs died before age 2 years, one of overwhelming sepsis and the other of cardiorespiratory failure associated with her cardiomyopathy. Metabolic studies showed a type 1 pattern of abnormal serum transferrin glycosylation. Fibroblasts synthesized truncated LLOs, primarily Man(7)GlcNAc(2), suggestive of CDG-Ig. Both sibs were compound heterozygotes for a novel 301 G > A (G101R) mutation and a previously described 437 G > A (R146Q) mutation in ALG12. Congenital disorders of glycosylation should be considered for children with undiagnosed multi-system disease including neurodevelopmental delay, skeletal dysplasia, immune deficiency, male genital hypoplasia, and cardiomyopathy.
Asunto(s)
Manosiltransferasas/deficiencia , Manosiltransferasas/genética , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/patología , Fenotipo , Agammaglobulinemia/patología , Enfermedades del Desarrollo Óseo/patología , Cardiomiopatías/patología , Análisis Mutacional de ADN , Resultado Fatal , Femenino , Genitales/anomalías , Glicosilación , Humanos , Recién Nacido , Lipopolisacáridos/metabolismo , Masculino , Mutación Missense/genética , Conformación Proteica , Transferrina/metabolismoRESUMEN
We describe a new Type II congenital disorder of glycosylation (CDG-II) caused by mutations in the conserved oligomeric Golgi (COG) complex gene, COG8. The patient has severe psychomotor retardation, seizures, failure to thrive and intolerance to wheat and dairy products. Analysis of serum transferrin and total serum N-glycans showed normal addition of one sialic acid, but severe deficiency in subsequent sialylation of mostly normal N-glycans. Patient fibroblasts were deficient in sialylation of both N- and O-glycans, and also showed slower brefeldin A (BFA)-induced disruption of the Golgi matrix, reminiscent of COG7-deficient cells. Patient fibroblasts completely lacked COG8 protein and had reduced levels and/or mislocalization of several other COG proteins. The patient had two COG8 mutations which severely truncated the protein and destabilized the COG complex. The first, IVS3 + 1G > A, altered the conserved splicing site of intron 3, and the second deleted two nucleotides (1687-1688 del TT) in exon 5, truncating the last 47 amino acids. Lentiviral-mediated complementation with normal COG8 corrected mislocalization of other COG proteins, normalized sialylation and restored normal BFA-induced Golgi disruption. We propose to call this new disorder CDG-IIh or CDG-II/COG8.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Mutación , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Transporte Biológico/efectos de los fármacos , Western Blotting , Brefeldino A/farmacología , Errores Innatos del Metabolismo de los Carbohidratos/genética , Errores Innatos del Metabolismo de los Carbohidratos/patología , Niño , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Glicosilación , Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi , Humanos , Lentivirus/genética , Masculino , Proteínas de la Membrana/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Transferrina/química , Transferrina/metabolismoRESUMEN
We describe a new cause of congenital disorder of glycosylation-Ic (CDG-Ic) in a young girl with a rather mild CDG phenotype. Her cells accumulated lipid-linked oligosaccharides lacking three glucose residues, and sequencing of the ALG6 gene showed what initially appeared to be a homozygous novel point mutation (338G>A). However, haplotype analysis showed that the patient does not carry any paternal DNA markers extending 33kb in the telomeric direction from the ALG6 region, and microsatellite analysis extended the abnormal region to at least 2.5Mb. We used high-resolution karyotyping to confirm a deletion (10-12Mb) [del(1)(p31.2p32.3)] and found no structural abnormalities in the father, suggesting a de novo event. Our findings extend the causes of CDG to larger DNA deletions and identify the first Japanese CDG-Ic mutation.
Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos/genética , Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/genética , Glucosiltransferasas/genética , Proteínas de la Membrana/genética , Trastornos Congénitos de Glicosilación/metabolismo , Femenino , Eliminación de Gen , Predisposición Genética a la Enfermedad/genética , Humanos , Recién Nacido , MutaciónRESUMEN
We present the clinical, molecular, and biochemical diagnosis of a patient with congenital disorder of glycosylation (CDG)-Ih. We report significant brain dysfunction in this multisystem disease, further expanding its complex clinical spectrum.
Asunto(s)
Enfermedades del Sistema Nervioso Central , Trastornos Congénitos de Glicosilación , Hepatopatías , Enteropatías Perdedoras de Proteínas , Enfermedades Óseas Metabólicas , Catarata , Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/metabolismo , Resultado Fatal , Humanos , Recién NacidoRESUMEN
In this report we describe the first two US patients with congenital disorder of glycosylation type Ig (CDG-Ig). Both patients presented with symptoms indicating CDG, including developmental delay, hypotonia and failure to thrive, and tested positive for deficient glycosylation of transferrin. Labeling of the patients' lipid-linked oligosaccharides suggested mutations in the hALG12 gene, encoding a mannosyltransferase. Both patients were shown to carry previously unpublished hALG12-mutations. Patient 1 has one allele with a deletion of G29, resulting in a premature stop codon, and another allele with an 824G>A mutation yielding an S275N amino acid change. Patient 2 carries two heterozygous mutations (688T>G and 931C>T), resulting in two amino acid exchanges, Y230D and R311C. An adenoviral vector expressing wild type hALG12 corrects the abnormal lipid-linked oligosaccharide pattern of the patients' cells. In addition to common CDG symptoms, these patients also presented with low IgG and genital hypoplasia, symptoms previously described in CDG-Ig patients. We therefore conclude that a combination of developmental delay, low IgG, and genital hypoplasia should prompt CDG testing.
Asunto(s)
Anomalías Múltiples/patología , Trastornos Congénitos de Glicosilación/genética , Inmunoglobulina G/metabolismo , Manosiltransferasas/genética , Adenoviridae , Células Cultivadas , Trastornos Congénitos de Glicosilación/patología , Cartilla de ADN , Prueba de Complementación Genética , Vectores Genéticos/genética , Glicosilación , Humanos , Recién Nacido , Masculino , Mutación Missense/genética , Oligosacáridos/metabolismo , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Estados UnidosRESUMEN
Congenital disorder of glycosylation (CDG) type Ic, the second largest subtype of CDG, is caused by mutations in human ALG6 (hALG6). This gene encodes the alpha1,3-glucosyltransferase that catalyzes transfer of the first glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. In this report, we describe the first adult patient diagnosed with CDG-Ic, carrying two previously unknown mutations. The first is a three base deletion (897-899delAAT) leading to the loss of I299, the second is an intronic mutation (IVS7 + 2T > G) that causes aberrant splicing. Wildtype hALG6, delivered by a lentiviral vector into patient's fibroblasts, clearly improves the biochemical phenotype, which confirms that the mutations are disease-causing. Striking clinical findings include limb deficiencies in the fingers, resembling brachydactyly type B, a deep vein thrombosis, pseudotumor cerebri, and endocrine disturbances with pronounced hyperandrogenism and virilization. However, even in adulthood, this patient shows normal magnetic resonance imaging of the brain.
Asunto(s)
Trastornos Congénitos de Glicosilación/patología , Glucosiltransferasas/genética , Proteínas de la Membrana/genética , Mutación , Adulto , Secuencia de Bases , Trastornos Congénitos de Glicosilación/genética , Análisis Mutacional de ADN , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Vectores Genéticos/genética , Glucosiltransferasas/metabolismo , Glicosilación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Ionización de Electrospray , TransfecciónRESUMEN
Adeno-associated virus (AAV) is a useful vector for hemophilia gene therapy, but the limited effective packaging capacity of AAV (5 kb) appears to be incompatible with factor VIII (gene symbol F8) cDNA (7 kb). Although we previously demonstrated efficient packaging and expression of B-domain-deleted human F8 (BDD-F8) using a single AAV vector, the packaging limit still excludes the use of large/strong regulatory elements. Here we exploited the split AAV vector technology that expands the packaging capacity of AAV through head-to-tail dimerization. To test the feasibility of AAV heterodimerization for F8 expression, we generated a 5' vector that includes a large enhancer/promoter cassette linked with exons 1-12 of the F8 cDNA and a half-intron-carrying splice donor site. A complementing 3' vector contains another half-intron-carrying splice acceptor site linked with the remaining F8 cDNA and a polyadenylation signal. Following coinfection of 293 and HepG2 cells, the 5' and 3' vectors together produced functional human factor VIII protein at a level of 120 mU/ml (24 ng/ml). No factor VIII protein was detected if only one of the vectors was used. Correct head-to-tail vector dimerization as well as spliced BDD-F8 mRNA was detected by DNA PCR and RT-PCR, respectively. Furthermore, intraportal injection of two rAAV/F8 vectors in immunodeficient mice produced 2% of the normal level of factor VIII for four months. Our results demonstrate the potential use of AAV dimerization for F8 expression.