A Novel Retrograde AAV Variant for Functional Manipulation of Cortical Projection Neurons in Mice and Monkeys / 神经科学通报·英文版

Retrograde adeno-associated viruses (AAVs) are capable of infecting the axons of projection neurons and serve as a powerful tool for the anatomical and functional characterization of neural networks. However, few retrograde AAV capsids have been shown to offer access to cortical projection neurons across different species and enable the manipulation of neural function in non-human primates (NHPs). Here, we report the development of a novel retrograde AAV capsid, AAV-DJ8R, which efficiently labeled cortical projection neurons after local administration into the striatum of mice and macaques. In addition, intrastriatally injected AAV-DJ8R mediated opsin expression in the mouse motor cortex and induced robust behavioral alterations. Moreover, AAV-DJ8R markedly increased motor cortical neuron firing upon optogenetic light stimulation after viral delivery into the macaque putamen. These data demonstrate the usefulness of AAV-DJ8R as an efficient retrograde tracer for cortical projection neurons in rodents and NHPs and indicate its suitability for use in conducting functional interrogations.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo / 中西医结合学报

OBJECTIVE@#Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy.@*METHODS@#The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.@*RESULTS@#rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.@*CONCLUSION@#Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Prokaryotic expression of a recombinant protein of adeno-associated virus capsid conserved regions and preparation of its polyclonal antibody / 南方医科大学学报

OBJECTIVE@#To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody.@*METHODS@#The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay.@*RESULTS@#The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10.@*CONCLUSION@#We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.

Co-editing PINK1 and DJ-1 Genes Via Adeno-Associated Virus-Delivered CRISPR/Cas9 System in Adult Monkey Brain Elicits Classical Parkinsonian Phenotype / 神经科学通报·英文版

Whether direct manipulation of Parkinson's disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6-10 months), and thus provides a practical transgenic monkey model for future PD studies.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy / 中西医结合学报

OBJECTIVE@#Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells.@*METHODS@#A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined.@*RESULTS@#The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.@*CONCLUSION@#HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Expression pattern of different serotypes of adeno-associated viral vectors in mouse retina / 北京大学学报(医学版)

OBJECTIVE@#To investigate the expression efficiency of exogenous gene mediated by different serotypes of adeno-associated virus (AAV) vectors in retina, and to compare the expression efficiency of AAV vector and two kinds of promoters commonly used in ophthalmology after transfection into mouse retina, so as to provide the basis for selecting appropriate AAV vector and promoter for gene therapy of retinitis pigmentosa.@*METHODS@#AAV2/2, AAV2/5, AAV2/8 and AAV2/9 were prepared. The C57BL/6J mice were injected subretinally with 1 μL purified AAV vectors (1.00×1013 mg/L). Then the mice were killed 2 or 4 weeks after treatment, and the eyes were enucleated for frozen section. The expression of green fluorescent protein (GFP) was observed under the confocal microscope. Two kinds of promoters, CMV and CAG, were selectd, and the expression of AAV2/8-GFP-CMV and AAV2/8-GFP-CAG was observed under confocal microscope.@*RESULTS@#No bacterial infection or immune response were seen in the injected mice. 2 weeks after injection, the GFP green fluorescence of AAV2/8 and AAV2/9 in the mouse retina was obvious, which indicated that the GFP green fluorescence of AAV2/8 and AAV2/9 was high after transfection into the mouse retina. In these two serotypes, GFP green fluorescence of AAV2/8 was mainly concentrated in photoreceptor cells while AAV2/8 was expressed in the whole retina, indicating that AAV2/8 was more specific to photoreceptors. Further experiments on AAV2/8 showed that the GFP green fluorescence of the mouse retina was obvious 4 weeks after injection, indicating that the exogenous gene mediated by AAV2/8 could be stably expressed in vivo. For CMV and CAG promoters, CMV promoter was expressed stronger in retinal pigment epithelium (RPE)cells, while CAG promoter was stronger in photorecepters. In photorecepters, CAG promoter was expressed almost the same as CMV promoter, while CMV promoter was stronger in RPE cells.@*CONCLUSION@#AAV vectors could express transgene robustly in retinal cells; Among several AAV serotypes, AAV2/2 and AAV2/5 showed weaker GFP fluorescence than AAV2/8 and AAV2/9. AAV2/9 showed expression in each layer of the retina including ganglion cells. AAV2/8 was more specific for photoreceptor; CAG promoters had higher specificity for photoreceptors than CMV promoters.

Respuesta inmune y terapia gó©nica con vectores virales adenoasociados / Immune response and gene therapy with adenoassociated viral vectors

En los ó;ºltimos aó;±os la terapia gó;©nica se ha posicionado como una opció;n real y segura en el desarrollo de alternativas terapó;©uticas para la cura y la prevenció;n de diferentes enfermedades. Consiste en la inserció;n de material genó;©tico en un tejido o có;©lula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unió;n y entrada a la có;©lula diana, la capacidad de transferencia del material genó;©tico al nó;ºcleo, la habilidad de expresió;n del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluació;n como opció;n terapó;©utica en un amplio nó;ºmero de enfermedades monogó;©nicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducció;n del vector e impiden la expresió;n del gen que transporta, limitando su aplicació;n clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia gó;©nica con vectores AAV. La presencia de NAb depende principalmente de la exposició;n al virus en la naturaleza y varía drásticamente segó;ºn edad, localizació;n geográfica y estado de salud de la persona evaluada.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient’s immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

Neurogenesis and gliogenesis modulation in cerebral ischemia by CDK5 RNAi-based therapy / Modulación de la neurogénesis y la gliogénesis en la isquemia cerebral mediante una terapia basada en la interferencia de CDK5

Abstract Introduction: Cerebral ischemia is the third cause of death risk in Colombia and the first cause of physical disability worldwide. Different studies on the silencing of the cyclin-dependent kinase 5 (CDK5) have shown that reducing its activity is beneficial in ischemic contexts. However, its effect on neural cell production after cerebral ischemia has not been well studied yet. Objective: To evaluate CDK5 silencing on the production of neurons and astrocytes after a focal cerebral ischemia in rats. Materials and methods: We used 40 eight-week-old male Wistar rats. Both sham and ischemia groups were transduced at CA1 hippocampal region with an adeno-associated viral vector using a noninterfering (shSCRmiR) and an interfering sequence for CDK5 (shCDK5miR). We injected 50 mg/kg of bromodeoxyuridine intraperitoneally from hour 24 to day 7 post-ischemia. We assessed the neurological abilities during the next 15 days and we measured the immunoreactivity of bromodeoxyuridine (BrdU), doublecortin (DCX), NeuN, and glial fibrillary acid protein (GFAP) from day 15 to day 30 post-ischemia. Results: Our findings showed that CDK5miR-treated ischemic animals improved their neurological score and presented increased BrdU+ cells 15 days after ischemia, which correlated with higher DCX and lower GFAP fluorescence intensities, and, although mature neurons populations did not change, GFAP immunoreactivity was still significantly reduced at 30 days post-ischemia in comparison with untreated ischemic groups. Conclusion: CDK5miR therapy generated the neurological recovery of ischemic rats associated with the induction of immature neurons proliferation and the reduction of GFAP reactivity at short and longterm post-ischemia.

Resumen Introducción. La isquemia cerebral es la tercera causa de riesgo de muerte en Colombia y la primera causa de discapacidad física en el mundo. En diversos estudios en los que se silenció la cinasa 5 dependiente de la ciclina (CDK5) se ha demostrado que la reducción de su actividad es beneficiosa frente a la isquemia. Sin embargo, su efecto sobre la neurogénesis después de la isquemia no se ha dilucidado suficientemente. Objetivo. Evaluar el silenciamiento de la CDK5 en la neurogénesis y la gliogénesis después de la isquemia cerebral focal en ratas. Materiales y métodos. Se usaron 40 machos de rata Wistar de ocho semanas de edad. Los grupos de control y los isquémicos sometidos a transducción en la región del hipocampo CA1, se inyectaron intraperitonealmente por estereotaxia con 50 mg/kg de bromodesoxiuridina (BrdU) a partir de las 24 horas y hasta el día 7 después de la isquemia, con un vector viral asociado a adenovirus usando una secuencia no interferente (SCRmiR) y una interferente (CDK5miR). Se evaluó la capacidad neurológica durante los quince días siguientes y se detectó la capacidad de inmunorreacción para la BrdU, la proteína doblecortina (DCX), los núcleos neuronales (NeuN), y la proteína fibrilar acídica de la glía (Glial Fibrillary Acidic Protein, GFAP) a los 15 y 30 días de la isquemia. Resultados. Los animales isquémicos tratados con CDK5miR mejoraron su puntuación neurológica y presentaron un incremento de la BrdU+ a los 15 días de la isquemia, lo cual se correlacionó con una mayor intensidad de la DCX+ y una menor de la GFAP+. No hubo modificación de los NeuN+, pero sí una reducción significativa de la GFAP+ a los 30 días de la isquemia en los animales tratados comparados con los animales isquémicos no tratados. Conclusión. La terapia con CDK5miR generó la recuperación neurológica de ratas isquémicas asociada con la inducción de la neurogénesis y el control de la capacidad de reacción de la proteína GFAP a corto y largo plazo después de la isquemia.

Adeno-Associated Virus 2-Mediated Hepatocellular Carcinoma is Very Rare in Korean Patients

BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.

Simple Purification of Adeno-Associated Virus-DJ for Liver-Specific Gene Expression

Recombinant gene expression using adeno-associated viruses (AAVs) has become a valuable tool in animal studies, as they mediate safe expression of transduced genes for several months. The liver is a major organ of metabolism, and liver-specific expression of a gene can be an invaluable tool for metabolic studies. AAV-DJ is a recombinant AAV generated by the gene shuffling of various AAV serotypes and shares characteristics of AAV2 and AAV8. AAV-DJ contains a heparin-binding domain in its capsid, which suggests that a heparin column could be used for the purification of the AAV. Given that AAV-DJ has been only recently available, relatively little is known about the optimal preparation/purification and application of AAV-DJ. Here, we present a simple large-scale preparation method that can generate 3×10(13) viral particles for in vivo experiments and demonstrate liver-specific gene expression via systemic injection in mice.

Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.

Protective Effect of Recombinant Adeno-Associated Virus 2/8-Mediated Gene Therapy from the Maternal Hyperphenylalaninemia in Offsprings of a Mouse Model of Phenylketonuria

Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pah(enu2), by the AAV serotype 2-mediated gene delivery of a human PAH transgene. However, the therapeutic efficacy had been limited to only male PKU mice. In this study, we generated a pseudotyped recombinant AAV2/8-hPAH vector and infused it into female PKU mice through the hepatic portal vein or tail vein. Two weeks after injection, complete fur color change to black was observed in female PKU, as in males. The PAH activity in the liver increased to 65-70% of the wild-type activity in female PKU mice and to 90% in male PKU mice. Plasma phenylalanine concentration in female PKU mice decreased to the normal value. In addition, the offsprings of the treated female PKU mice can rescue from the harmful effect of maternal hyperphenylalaninemia. These results indicate that recombinant AAV2/8-mediated gene therapy is a potential therapeutic strategy for PKU.

Promoter effects of adeno-associated viral vector for transgene expression in the cochlea in vivo

The aims of this study were to evaluate the expression of enhanced green fluorescent protein (EGFP) driven by 6 different promoters, including cytomegalovirus IE enhancer and chicken beta-actin promoter (CAG), cytomegalovirus promoter (CMV), neuron-specific enolase promoter (NSE), myosin 7A promoter (Myo), elongation factor 1alpha promoter (EF-1alpha), and Rous sarcoma virus promoter (RSV), and assess the dose response of CAG promoter to transgene expression in the cochlea. Serotype 1 adeno-associated virus (AAV1) vectors with various constructs were transduced into the cochleae, and the level of EGFP expression was examined. We found the highest EGFP expression in the inner hair cells and other cochlear cells when CAG promoter was used. The CMV and NSE promoter drove the higher EGFP expression, but only a marginal activity was observed in EF-1alpha promoter driven constructs. RSV promoter failed to driven the EGFP expression. Myo promoter driven EGFP was exclusively expressed in the inner hair cells of the cochlea. When driven by CAG promoter, reporter gene expression was detected in inner hair cells at a dose as low as 3 x 10(7) genome copies, and continued to increase in a dose- dependent manner. Our data showed that individual promoter has different ability to drive reporter gene expression in the cochlear cells. Our results might provide important information with regard to the role of promoters in regulating transgene expression and for the proper design of vectors for gene expression and gene therapy.

A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound-healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.

Recombinant adeno-associated virus mediated gene transfer in a mouse model for homocystinuria

Homocystinuria is a metabolic disorder caused by a deficiency of cystathionine b-synthase (CBS). The major clinical symptoms of this disease are mental retardation, lens dislocation, vascular disease with life-threatening thromboembolisms, and skeletal deformities. The major treatments for CBS deficiency include pharmacologic doses of pyridoxine or dietary restriction of methionine. There is currently no effective long-term treatment to lower the elevated plasma levels of homocysteine. However, gene therapy could be an effective novel approach for the treatment of homocystinuria. A recombinant adeno- associated virus vector carrying human CBS cDNA (rAAV-hCBS) was constructed and administered to CBS-/- mice by intramuscular (IM) and intraperitoneal (IP) injections. Serum homocysteine concentrations significantly decreased in treated mice compared with age-matched controls two weeks after treatment. The treated CBS-/- mice had life spans 3-7 days longer compared with untreated CBS-/- mice. In CBS-/- mice treated with rAAV-hCBS via IP injection, the vector was detected in all organs examined including liver, spleen, and kidney, and CBS gene expression was observed by immunohistochemical staining in the liver. These results indicate the efficacy of gene delivery and demonstrate the possibility of gene therapy mediated by AAV gene transfer in this mouse model of homocystinuria.

Effects of rAAV-CD151 and rAAV-antiCD151 on the migration of human tongue squamous carcinoma cell line Tca8113 / 华中科技大学学报（医学）（英德文版）

This study was designed to determine the effects of the recombinant adeno-associated virus vector containing sense CD151 gene (rAAV-CD151) and antisense CD151 gene (rAAV-antiCD151) on the migration of Tca8113 cell. Functional fragment of CD151 gene was amplified by RT-PCR, and inserted into the vector pAAV in the sense direction and antisense direction, respectively. The rAAV-CD151 and rAAV-antiCD151 were produced and the titers were determined by dot blot. The CD151, at protein level, was detected by Western blot. The Transwell chamber was used to detect the effects of the rAAV-CD151 and rAAV-antiCD151 on the tumor cell migration. The titers of the rAAV-CD151 and rAAV-antiCD151 were 2 x 10(11) pfu/ml and 1.0 x 10(11) pfu/ml, respectively. The expression of CD151 was increased by 108% in the cells transfected with rAAV-CD151 and decreased by 79% in the cells transfected with rAAV-antiCD151, as compared with non-transfected cells, respectively. The number of the migrating cells was significantly increased in the cells transfected with rAAV-CD151 (93.56 +/- 11.59) and decreased in the cells transfected with rAAV-antiCD151 (24.00 +/- 4.36) as compared with non-transfected and rAAV-GFP transfected cells (53.00 +/- 6.56 and 46.00 +/- 7.00, P<0.05). It is an important molecular mechanism of the tumor metastasis that the overexpression of CD151 promotes the migration of the tumor cells. The rAAV-antiCD151 is a novel tool, which can reduce the expression of CD151 and inhibit the migration of the tumor cells, and brings us a new approach of anti-sene gene therapy targeted at CD151 in human carcinoma.