Biomedical subjects

W Xiao

Publications and source records attributed to W Xiao.

At least 73 records · Page 4Linked to original sources

PubMed2000

[Otogenic hypertrophic cranial pachymeningitis associated with edema of the temporal lobe and organic mental disorder-case report].

OBJECTIVE: Hypertrophic cranial pachymeningitis(HCP) is a rare disease which might be misdiagnosed. One typical case of otogenic hypertrophic cranial pachymeningitis (HCP) associated with edema of the temporal lobe and organic mental disorder was reported. Literatures associated with HCP were reviewed for reference. METHODS: In July, 1997, a 26-year-old man was admitted for right-sided severe headache, dizziness, nausea, vomiting, facial and abducens nerve palsy. MRI and CT revealed inflammation in the temporal bone and locally thickened dura mater with obvious enhancement as a band in the petrous apex and nearby tentorium cerebellum. The patient then underwent operation including decompression of the facial nerve, labyrinthectomy, and eradication of the inflammatory cells in the mastoid and petrous portion. Postoperatively, the clinical signs disappeared except hearing loss. Seven months later, the patient suffered from mental disorder with interrupted excitement to hit and abuse the family members. He was then treated in a psychosis hospital but no improvement could be seen. On May 13, 1998, MRI and the enhanced MRI demonstrated diffuse thickened tentorium cerebellum and dura mater in the middle cranial fossa. The inferior portion of the temporal lobe was obvious edema. The internal carotid artery was partially occluded due to the thickened wall of cavernous sinus. RESULTS: Hence, the diagnosis of HCP associated with edema of temporal lobe and organic mental disorder was established. The patient was cured by high dosage of penicillin. CONCLUSION: MRI is an essential method for diagnosis of HCP. Antibiotic was an effective treatment.

Adult↗

PubMed1999

Insertional mutagenesis of AAV2 capsid and the production of recombinant virus.

The structural genes of adeno-associated virus serotype 2 (AAV2) have been altered by linker insertional mutagenesis in order to define critical components of virion assembly and infectivity. An in-frame restriction site linker was inserted across the capsid coding domain of a recombinant plasmid. After complementation in vivo, recombinant AAV2 viruses were generated and assayed for capsid production, packaging, transduction, heparin agarose binding, and morphology. Three classes of capsid mutants where identified. Class I mutants expressed structural proteins but were defective in virion assembly. Class II mutants generated intact virions that protected the viral genome from DNase, but failed to infect target cells. The majority of these mutants bound the heparin affinity matrix, suggesting that attachment to the AAV primary receptor was not rate limiting. One class II mutant, H2634, assembled virions and bound heparin using only Vp3, indicating that this subunit is responsible for mediating AAV receptor attachment. Finally, class III mutants assembled virions, encapsidated DNA, and infected target cells. Infectivity of these mutants ranged from 5 to 100% of that of the wild-type, demonstrating for the first time the ability to alter capsid proteins without interfering with infectivity. These AAV virions with altered capsid subunits will provide critical templates for manipulating AAV vectors for cell-specific gene delivery in vivo. In summary, the AAV capsid variants described here will facilitate further study of virus assembly, entry, and infection, as well as advance the development of this versatile vector system.

Capsid↗

PubMed1999

REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase.

O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure. It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate. In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains. To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition. Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair. The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3. We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains. These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations. However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG.

Base Sequence↗

PubMed1999

Methionine reduces spontaneous and alkylation-induced mutagenesis in Saccharomyces cerevisiae cells deficient in O6-methylguanine-DNA methyltransferase.

The exposure of DNA to reactive intracellular metabolites is thought to be a major cause of spontaneous mutagenesis. DNA alkylation is implicated in the above process by the fact that bacterial and yeast cells lacking DNA alkylation-specific repair genes exhibit elevated spontaneous mutation rates. The origin of the intracellular alkylating molecules is not clear; however, S-adenosylmethionine (SAM) has been proposed as one source because it has a reactive methyl group known to methylate proteins and DNA. We supplemented yeast cultures with excess methionine and examined the effects of increased endogenous SAM concentration on spontaneous and alkylation-induced mutagenesis in the absence of various DNA repair pathways. Our results show that either the excess methionine, or the increased SAM produced as a result of this treatment, is able to protect yeast cells from mutagenesis, and that this effect is alkylation-damage-specific. The protective effect was observed only in the mgt1 mutant deficient in the O(6)-methylguanine-DNA repair methyltransferase, but not in the wild type or other DNA repair-deficient strains, indicating that the protection is specific for O-methyl lesions. Thus, our results may lend support to the recently reported chemopreventive effect of SAM in rodents and further suggest that the observed tumor prevention by SAM may be, in part, due to its suppression of spontaneous mutagenesis in mammals. Given that a strong correlation has been established between O(6)-methylguanine and carcinogenicity, this study may offer a novel approach to preventing carcinogenesis.

Alkylation↗

PubMed1999

Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae.

Evidence obtained from recent studies supports the existence of an error-free postreplication repair (PRR) and a mutagenesis pathway within the Saccharomyces cerevisiae RAD6 DNA repair group. The MMS2 gene is the only known yeast gene involved in error-free PRR that, when mutated, significantly increases the spontaneous mutation rate. In this study, the mutational spectrum of the mms2 mutator was determined and compared to the wild type strain. In addition, mutagenenic effects and genetic interactions of the mms2 mutator and rev3 anti-mutator were examined with respect to forward mutations, frameshift reversions as well as amber and ochre suppressions. It was concluded from these results that the mms2 mutator phenotype is largely dependent on the functional REV3 gene. The synergistic effects of mms2 and rev3 mutations towards killing by a variety of DNA-damaging agents ruled out the possibility that MMS2 simply acts to suppress REV3 activity and favored the hypothesis that MMS2 and REV3 form two alternative subpathways within the RAD6 DNA repair pathway. Taken together, we propose that two pathways represented by MMS2 and REV3 deal with a similar range of endogenous and environmental DNA damage but with different biological consequences, namely, error-free repair and mutagenesis, respectively.

Base Sequence↗

PubMed1999

A piston model for transmembrane signaling of the aspartate receptor.

To characterize the mechanism by which receptors propagate conformational changes across membranes, nitroxide spin labels were attached at strategic positions in the bacterial aspartate receptor. By collecting the electron paramagnetic resonance spectra of these labeled receptors in the presence and absence of the ligand aspartate, ligand binding was shown to generate an approximately 1 angstrom intrasubunit piston-type movement of one transmembrane helix downward relative to the other transmembrane helix. The receptor-associated phosphorylation cascade proteins CheA and CheW did not alter the ligand-induced movement. Because the piston movement is very small, the ability of receptors to produce large outcomes in response to stimuli is caused by the ability of the receptor-coupled enzymes to detect small changes in the conformation of the receptor.

Aspartic Acid↗

PubMed1999

Formation of the yeast Mre11-Rad50-Xrs2 complex is correlated with DNA repair and telomere maintenance.

The yeast Mre11 is a multi-functional protein and is known to form a protein complex with Rad50 and Xrs2. In order to elucidate the relationship between Mre11 complex formation and its mitotic functions, and to determine domain(s) required for Mre11 protein interactions, we performed yeast two-hybrid and functional analyses with respect to Mre11 DNA repair and telomere maintenance. Evidence presented in this study indicates that the N-terminal region of Mre11 constitutes the core homo-dimerization and hetero-dimerization domain and is sufficient for Mre11 DNA repair and maintaining the wild-type telomere length. In contrast, a stretch of 134 amino acids from the extreme C-terminus, although essential for achieving a full level of self-association, is not required for the aforementioned Mre11 mitotic functions. Interestingly, deletion of these same 134 amino acids enhanced the interaction of Mre11 with Rad50 and Xrs2, which is consistent with the notion that this region is specific for meiotic functions. While Mre11 self-association alone is insufficient to provide the above mitotic activities, our results are consistent with a strong correlation between Mre11-Rad50-Xrs2 complex formation, mitotic DNA repair and telomere maintenance. This correlation was further strengthened by analyzing two mre11 phosphoesterase motif mutants ( mre11-2 and rad58S ), which are defective in DNA repair, telomere maintenance and protein interactions, and a rad50S mutant, which is normal in both complex formation and mitotic functions. Together, these results support and extend a current model regarding Mre11 structure and functions in mitosis and meiosis.

Base Sequence↗

PubMed1999

Microemulsion of seal oil markedly enhances the transfer of a hydrophobic radiopharmaceutical into acetylated low density lipoprotein.

Four different microemulsions differing in their core lipid component (triolein, canola oil, squalene, or seal oil) and containing 1,3-dihydroxypropan-2-one 1,3-diiopanoate (DPIP), a potential radioimaging probe, were prepared by means of ultrasonication. The DPIP microemulsions were incubated with acetylated human low density lipoprotein (AcLDL) and the amount of DPIP transferred into AcLDL was examined. The amount of DPIP in the microemulsions expressed as DPIP/oil (w/w) was dependent on the core lipid component of the microemulsion in the order of seal oil (0.19+/-0.04, mean +/- standard deviation) > squalene (0.15+/-0.02) > canola oil (0.12+/-0.02) > triolein (0.07+/-0.004). With the exception of canola oil, all microemulsions were effective in enhancing the transfer of DPIP into AcLDL in comparison with commonly used methods, i.e., direct diffusion and detergent solubilization. DPIP in seal oil resulted in the highest amount of DPIP transferred into AcLDL [309.16+/-34.82 vs. 203.19+/-64.51 using squalene and 151.31+/-28.54 using triolein (DPIP molecules per AcLDL particle)]. For the first time, oil from harp seals, was studied as a major core lipid component of formulating pharmaceutical microemulsions. DPIP in seal oil resulted in the highest transfer of DPIP into AcLDL which is likely due to the highest DPIP concentration found in this microemulsion as well as the high fluidity of seal oil.

Animals↗

PubMed1999

2-5A-DNA conjugate inhibition of respiratory syncytial virus replication: effects of oligonucleotide structure modifications and RNA target site selection.

To define more fully the conditions for 2-5A-antisense inhibition of respiratory syncytial virus (RSV), relationships between 2-5A antisense oligonucleotide structure and the choice of RNA target sites to inhibition of RSV replication have been explored. The lead 2-5A-antisense chimera for this study was the previously reported NIH8281 that targets the RSV M2 RNA. We have confirmed and extended the earlier study by showing that NIH8281 inhibited RSV strain A2 replication in a variety of antiviral assays, including virus yield reduction assays performed in monkey (EC90 = 0.02 microM) and human cells (EC90 = microM). This 2-5A-antisense chimera also inhibited other A strains, B strains and bovine RSV in cytopathic effect inhibition and Neutral Red Assays (EC50 values = 0.1-1.6 microM). The 2'-O-methylation modification of NIH8281 to increase affinity for the complementary RNA and provide nuclease resistance, the introduction of phosphothioate groups in the antisense backbone to enhance resistance to exo- and endonucleases, and the addition of cholesterol to the 3'-terminus of the antisense oligonucleotide to increase cellular uptake, all resulted in loss of activity. Of the antisense chimeras targeting other RSV mRNAs (NS1, NS2, P, M. G, F, and L), only those complementary to L mRNA were inhibitory. These results suggest that lower abundance mRNAs may be the best targets for 2-5A-antisense; moreover, the active 2-5A antisense chimeras in this study may serve as useful guides for the development of compounds with improved stability, uptake and anti-RSV activity.

Adenine Nucleotides↗

PubMed1999

Radiolabeled cholesteryl iopanoate/acetylated low density lipoprotein as a potential probe for visualization of early atherosclerotic lesions in rabbits.

PURPOSE: Atherosclerosis is the underlying factor leading to such cardiovascular diseases (CVD) as stroke, aneurysm, and myocardial infarction. The early detection of atherosclerotic plaques is considered to be crucial for successful prevention and/or therapeutic and dietary intervention of CVD. Current diagnostic practice, on the other hand, can only detect the problem at an advanced stage. The purpose of this study was to examine the potential of using a radiolabeled cholesterol ester analog/acetylated low density lipoprotein (AcLDL) conjugate as a diagnostic agent for the early and non-invasive detection of atherosclerosis and for the monitoring of the effects of drug therapy. METHODS: Cholesteryl iopanoate (CI), a cholesterylester analog, was synthesized, radiolabeled, and incorporated into AcLDL. Early atherosclerotic lesions were induced in New Zealand White rabbits. 125[-CI/AcLDL was injected intravenously at 2 microCi/kg. Blood samples were taken at different time intervals after injection and clearance of the injected drug from blood was studied. The rabbits were sacrificed after 72 hours and the distribution of radioactivity in various organs was investigated. Aortae of both atherosclerotic lesion and control rabbits were removed for Sudan IV staining and autoradiography in order to confirm the formation of the atherosclerotic lesion and localization of radioactivity. RESULTS: The injected drug was found to be cleared from blood following a two compartment model. Radioactivity in the atherosclerotic aorta was found to be about 8 times higher than that in normal aorta, suggesting that the proposed diagnostic probe was selectively taken up by the atherosclerotic lesion. The autoradiography and staining confirmed that the localization of the proposed probe was superimposed with the atherosclerotic lesion site. CONCLUSIONS: The results suggested that incorporation of CI into AcLDL resulted in the selective localization of CI at the atherosclerotic plaque areas. CI/AcLDL labeled with appropriate radioisotope has the potential to be used as a probe for visualization of early atherosclerotic lesion using scintigraphy technology.

Animals↗

PubMed1999

Charge-to-alanine mutagenesis of the adeno-associated virus type 2 Rep78/68 proteins yields temperature-sensitive and magnesium-dependent variants.

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32 degrees C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37 degrees C and was undetectable at 39 degrees C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting a ts phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 x 10(6) and 3 x 10(3), respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg(2+)-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep ts mutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.

Alanine↗

PubMed1999

Gene therapy vectors based on adeno-associated virus type 1.

The complete sequence of adeno-associated virus type 1 (AAV-1) was defined. Its genome of 4,718 nucleotides demonstrates high homology with those of other AAV serotypes, including AAV-6, which appears to have arisen from homologous recombination between AAV-1 and AAV-2. Analysis of sera from nonhuman and human primates for neutralizing antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i) NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates, while the reverse is true in humans; and (ii) sera from 36% of nonhuman primates neutralized AAV-1 but not AAV-2, while sera from 8% of humans neutralized AAV-2 but not AAV-1. An infectious clone of AAV-1 was isolated from a replicated monomer form, and vectors were created with AAV-2 inverted terminal repeats and AAV-1 Rep and Cap functions. Both AAV-1- and AAV-2-based vectors transduced murine liver and muscle in vivo; AAV-1 was more efficient for muscle, while AAV-2 transduced liver more efficiently. Strong NAB responses were detected for each vector administered to murine skeletal muscle; these responses prevented readministration of the same serotype but did not substantially cross-neutralize the other serotype. Similar results were observed in the context of liver-directed gene transfer, except for a significant, but incomplete, neutralization of AAV-1 from a previous treatment with AAV-2. Vectors based on AAV-1 may be preferred in some applications of human gene therapy.

Adolescent↗

PubMed1999

Transduction of well-differentiated airway epithelium by recombinant adeno-associated virus is limited by vector entry.

The limitations of adeno-associated virus (AAV)-mediated vectors for lung-directed gene transfer were investigated by using differentiated human respiratory epithelium in air-liquid interface cultures. Transduction efficiency was high in undifferentiated cells and was enhanced in well-differentiated cells after basolateral application of the vector or after apical application following disruption of tight junctions or pretreatment of the cultures with glycosidases. These results indicate that transduction of airway epithelia by AAV vectors is limited by entry and reinforce the importance of a physical barrier on the airway surface.

Cell Differentiation↗

PubMed1999

Incorporation of an (125)I-labeled hexa-iodinated diglyceride analog into low-density lipoprotein and high specific uptake by cells of cervical carcinoma cell lines.

The feasibility of using low-density lipoprotein (LDL) to deliver cytotoxic drugs to tumor cells has been explored since the 1980s, when cells of a number of cancer cell lines were found to have higher LDL receptor activity than normal cells. Such differential uptake between tumor and normal cells may provide a unique opportunity to use LDL as a tumor-specific carrier of radiopharmaceuticals for the clinical management of cancer. In this study, an (125)I-labeled hexa-iodinated diglyceride analog, 1, 3-dihydroxypropan-2-one 1,3-diiopanoate (DPIP), was synthesized and incorporated into LDL using a fusion technique. It was found that approximately 500 [(125)I] DPIP molecules were incorporated into each LDL particle. Cells of three human cervical tumor cell lines, HeLa, SiHa and C-33A, were used to examine the cellular uptake of the [(125)I]DPIP-LDL conjugate. It was shown that the [(125)I]DPIP-LDL conjugate was specifically bound to and taken up by cervical tumor cells through an LDL receptor-mediated endocytosis pathway. The results suggest that LDL may be a selective carrier for delivering hydrophobic radiopharmaceuticals to cancer cells and particularly for the diagnosis of cervical tumors.

Binding Sites↗

PubMed1998

Isolation of hMRE11B: failure to complement yeast mre11 defects due to species-specific protein interactions.

The Saccharomyces cerevisiae MRE11 gene plays an important role in meiotic recombination, mitotic DNA repair and telomere maintenance. We present the isolation of hMRE11B cDNA from a human HeLa cell cDNA library as an MRE11 homolog. Compared to the previously identified hMRE11, hMRE11B contains an additional 84bp sequence that results in a 28 amino-acid insertion close to the C-terminus. The expression pattern of hMRE11B in different tissues shows the presence of two mRNA species of approx. 2.6 and 7.5kb. Overexpression of hMRE11B does not complement the alkylation sensitivity of the mre11 null and temperature-sensitive mutant strains. In this study, we examine factors that may explain this lack of complementation. First, both Northern and Western analyses rule out the lack of hMRE11B transcription and/or translation in yeast. Second, we demonstrate that hMre11B, like the yeast Mre11 protein, dimerizes in vivo in a yeast two-hybrid system. This dimerization requires the C-terminal one-third of hMre11B protein, which includes the 28 amino acids absent in hMre11. However, hMre11B does not interact with Mre11, Rad50 and Xrs2. Hence, the lack of protein-protein interaction between hMre11B and the yeast Mre11, Rad50, and Xrs2 may explain the inability of hMRE11B to complement the yeast mre11 mutants. We rule out the hypothesis that the lack of interaction and, in turn of complementation, is due to the absence of sequence homology at the C-terminal domain of hMre11B compared to the yeast Mre11. Instead, we propose that the C-terminus of hMre11B participates in protein-protein interaction and functions in a species-specific manner.

Base Sequence↗

PubMed1998

Differential regulation of two closely clustered yeast genes, MAG1 and DDI1, by cell-cycle checkpoints.

Eukaryotic DNA-damage checkpoint genes have been shown to not only arrest cells at certain stages, but are also involved in the transcriptional response to DNA damage. However, while the signal transduction for cell-cycle checkpoint is well characterized, it is not clear whether the same signal transduction pathway is responsible for the regulation of all DNA damage-inducible genes. In order to understand how different checkpoint genes are involved in gene regulation, the effects of various checkpoint mutations on the expression of a unique yeast MAG1 - DDI1 dual promoter were examined in this study. MAG1 and DDI1 are transcribed from a common promoter region and co-induced by a variety of DNA damaging agents. However, gene-specific cis -acting elements were also identified, and the two genes are indeed differentially expressed under certain conditions. We found that DDI1 induction was not affected in any of the checkpoint mutants. In contrast, MAG1 induction was completely abolished in the pol2 and rad53 mutants. However, in the mec1-1 or any of the G1/S and G2/M checkpoint mutants, including rad9, rad17 and rad24, DNA damage-induced MAG1 expression was not significantly affected, and a rad9 rad17 double mutation only slightly reduced MAG1 induction. Based on this and previous studies, we present two models for the role of checkpoint genes in transcriptional regulation in response to DNA damage.

Cell Cycle↗

PubMed1998

2',5'-Oligoadenylate-antisense chimeras cause RNase L to selectively degrade bcr/abl mRNA in chronic myelogenous leukemia cells.

We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2', 5'-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210(bcr/abl) kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of beta-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.

Adenine Nucleotides↗

PubMed1998

High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus.

Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.

Animals↗