Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.

We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p40. This promoter is embedded within the carboxyl-terminal region of an open reading frame (orf-1) which codes for a protein (rep) required for AAV DNA replication. We show here that the rep product has additional trans-acting properties to regulate gene expression. First, deletion or frame-shift mutations in orf-1, which occurred far upstream of p40, increased expression of CAT in human 293 (adenovirus-transformed) cells. This increased CAT expression was abolished when such mutant AAV vectors were transfected into 293 cells together with a second AAV vector which could supply the wild-type AAV rep product in trans. Thus, an AAV rep gene product was a negative regulator, in trans, of expression of CAT in uninfected 293 cells. In adenovirus-infected 293 cells, the function of the AAV rep product was more complex, but in some cases, it appeared to be a trans activator of the expression from p40. In HeLa cells, only trans activation by rep was seen in the absence or presence of adenovirus. Neither activation nor repression by the rep product required replication per se of the AAV vector DNA. Thus, trans-acting negative or positive regulation of gene expression by the AAV rep gene is modulated by factors in the host cell and by the helper adenovirus.

Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells.

We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.

A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase.

We have used the defective human parvovirus adeno-associated virus (AAV) as a novel eucaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p40 (pAVHiCAT) and p19 (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p19 is increased by E1A, whereas p40 yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

A mutant cell line derived from NIH/3T3 cells: two oncogenes required for in vitro transformation.

EK-3, a cell line derived from NIH/3T3 cells, was isolated. These cells are nontumorigenic to NIH Swiss nude mice. They required both myc and ras genes for in vitro transformation in contrast to NIH/3T3 cells, which are efficiently transformed following transfection by ras alone. Two other genes, chloramphenicol acetyl transferase and geneticin resistance, could be efficiently transfected and expressed in both EK-3 cells and the parental NIH/3T3 cells. Thus the possibility that the requirement of myc in EK3 cells is due to low efficiency of transfection could be ruled out. The present study suggests that myc plays a significant role in the overall process of transformation rather than simply immortalization of the cells. The EK-3 line can be very helpful in elucidating this function.

Inhibition of adenovirus oncogenicity in hamsters by adeno-associated virus DNA.

Adeno-associated virus (AAV) DNA partially inhibited in Syrian golden hamsters the formation of tumors by the human adenovirus (Ad) type 12. Furthermore, defective interfering AAV particles or their DNA also reduced tumorigenesis. Defective AAV particles contain aberrant genomes with extensive deletions of the internal AAV DNA sequences. Variant AAV DNA, containing 30% of the viral genome, decreased the incidence of Ad-induced tumors from 44 to 18%. Defective AAV particles, which have a buoyant density of 1.32 g/cm3 in CsCl and which are highly enriched for the DNA of the terminal regions (map positions, 0-5 and 95-100), completely suppressed Ad oncogenicity. This observation suggested that the AAV DNA sequences close to the terminal region of the genome mediated the inhibition of the Ad oncogenicity.

Effect of adeno-associated virus on transformation of NIH 3T3 cells by ras gene and on tumorigenicity of an NIH 3T3 transformed cell line.

Transfection of NIH-3T3 cells with the plasmid pJ234, containing DNA from the human bladder carcinoma T24 cell line (ras gene), results in their transformation. Adeno-associated virus did not affect significantly the number of the transformed foci when different multiplicities of infection were used and when the virus was added to the cultures at different time intervals before or after transfection. A transformed cell line was derived following transfection of NIH 3T3 cells by the ras gene. Infection of these cells with adeno-associated virus resulted in a decrease in their growth rate and cloning efficiency. These infected cells showed a dose-dependent reduction in the frequency and an increase in the latent period for tumor appearance in nude mice.

Replication of adeno-associated virus DNA. Complementation of naturally occurring rep- mutants by a wild-type genome or an ori- mutant and correction of terminal palindrome deletions.

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.

Adeno-associated virus vectors.

Adeno-associated virus is a human parvovirus that integrates its DNA genome into host cell chromosomes with very high efficiency. This suggests that adeno-associated virus may be a useful vector for human gene therapy. Interest in adeno-associated virus vectors increased greatly in the last year following reports that adeno-associated virus genome integration may be site specific and occur at preferred sites in the human genome. Several genes relevant to the treatment of genetic or infectious diseases have been expressed in adeno-associated virus vectors in vitro.

Cloning of the human thyrotropin beta-subunit gene and transient expression of biologically active human thyrotropin after gene transfection.

A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human alpha- and TSH beta-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunoradiometric and in vitro iodide trapping assay, respectively.

A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease.

Cystic fibrosis (CF) is one of the most common autosomal recessive disorders in North America, leading to significant morbidity and early mortality. The defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) function can be corrected in vitro by gene replacement with a wild-type gene. A Phase I, single administration, dose escalation trial was designed and executed to assess safety and delivery of tgAAVCF, an adeno-associated virus (AAV) vector encoding the human CFTR cDNA, by nebulization to the lungs of CF subjects. Four cohorts of three subjects each were administered increasing doses of the study agent, beginning with 10(10) DNase-resistant particles (DRP) and escalating in log increments up to 10(13) DRP. Sequential bronchoscopies were performed to gather analytical samples throughout the study. All 12 subjects completed the study. There were a total of 242 adverse events (AEs), six of which were defined as serious and three of which were defined as possibly being related to the study drug. A clear dose-response relationship was observed in vector gene transfer. A maximum of 0.6 and 0.1 vector copies per brushed cell were observed 14 days and 30 days, respectively, following nebulization of 10(13) DRP tgAAVCF, and this declined to nearly undetectable levels by day 90. Vector gene transfer was evenly distributed throughout the fourth airway generation following single-dose administration. RNA-specific PCR did not detect vector-derived mRNA. This Phase I trial shows that aerosolized tgAAVCF is safe and widely delivered to the proximal airways of CF subjects by nebulization.

Cloning of infectious adeno-associated virus genomes in bacterial plasmids.

We describe the construction of two Escherichia coli hybrid plasmids, each of which contains the entire 4.7-kb DNA genome of the human parvovirus, adeno-associated virus (AAV) type 2. Because the AAV genome was inserted into the plasmid DNA using BglII linkers the entire virus genome can be recovered by in vitro cleavage of the purified recombinant plasmid. Transfection of these recombinant DNAs into an adenovirus-transformed human cell line in the presence of helper adenovirus resulted in efficient rescue and replication of the AAV genome and production of fully infectious virus particles. These AAV-plasmid recombinant DNA molecules should be useful both for site-specific mutagenesis of the viral genome and to study the potential of AAV as a eukaryotic vector.

A lambda library of Herpes simplex virus type 1 (KOS) DNA fragments obtained by partial digestion with Sau3A.

Large fragments of Herpes simplex virus type 1 (HSV-1, strain KOS) DNA were produced by partial cleavage with Sau3A and inserted into a phage lambda BamHI vector. Recombinant phage (lambda KOS) DNA molecules were isolated and characterised. The final collection of phage recombinants contains partially overlapping inserts, which represent most of the HSV-1 genome. Restriction enzyme analysis of many independent clones containing Us sequences revealed sequence polymorphism in two specific regions.

Purification of epithelial-type transforming growth factor by micro-preparative electrophoresis chromatography.

The purification and recovery of biologically active epithelial-type transforming growth factor (TGFe) is described. In the final phase of purification, micropreparative electrophoretic chromatography was employed using a Tris-glycine-sodium dodecyl sulfate buffer system in an automated instrument. Briefly, partially purified protein preparations were separated in 2.5 x 50 mm, 10% polyacrylamide gel in electrophoresis tubes installed in the apparatus, electrophoresed under constant current of 1.5 mA for 400 min and recovered by automated fractionation and collection of the eluant from the tube gel. Aliquots of the eluted fractions were assayed in a biological system using SW-13 cell growth stimulation as an indicator of the presence of biologically active TGFe. Using the above procedure, TGFe was purified to within 95% homogeneity as assessed by silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Treatment of childhood obesity in pediatric practice.

Evaluation of obese children and adolescents in the pediatric office or clinic should include baseline assessment of weight for height, and body fatness; should rule out endocrine and genetic causes of obesity; and should evaluate other cardiovascular risk factors. Treatment of obesity is most successful if realistic goals are set; if a safe rate of weight loss of one to two pounds per week can be achieved through a reduction of caloric intake that amounts to 500 calories less per day; if increased physical activity is stressed as much as diet; if parental support is strong; and if behavior therapy is provided during the course of treatment to help both child and parent achieve the diet, exercise, and behavior goals.

Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus.

OBJECTIVE: The host immune response and low vector efficiency have been key impediments to effective cystic fibrosis transmembrane regulator (CFTR) gene transfer for cystic fibrosis (CF). An adeno-associated virus vector (AAV-CFTR) was used in a phase I dose-escalation study to transfer CFTR cDNA into respiratory epithelial cells of the maxillary sinus of 10 CF patients. STUDY DESIGN: A prospective, randomized, unblinded, dose-escalation, within-subjects, phase I clinical trial of AAV-CFTR was conducted. PATIENTS: Ten patients with previous bilateral maxillary antrostomies were treated. MAIN OUTCOME MEASURES: Safety, gene transfer as measured by semiquantitative polymerase chain reaction (PCR), and sinus transepithelial potential difference (TEPD) were measured. RESULTS: The highest level of gene transfer was observed in the range of 0.1-1 AAV-CFTR vector copy per cell in biopsy specimens obtained 2 weeks after treatment. When tested, persistence was observed in one patient for 41 days and in another for 10 weeks. Dose-dependent changes in TEPD responses to pharmacologic intervention were observed following treatments. Little or no inflammatory or immune responses were observed. CONCLUSION: AAV-CFTR administration to the maxillary sinus results in successful, dose-dependent gene transfer to the maxillary sinus and alterations in sinus TEPD suggestive of a functional effect, with little or no cytopathic or host immune response. Further study is warranted for AAV vectors as they may prove useful for CFTR gene transfer and other in vivo gene transfer therapies. A prospective, randomized, double-blind, placebo-controlled, within-subjects, phase II clinical trial of the effect AAV-CFTR on clinical recurrence of sinusitis will determine the clinical efficacy of AAV gene therapy for CF.

Long-term survivors of breast cancer. A qualitative descriptive study.

The purpose of this study was to explore the daily lived experience of women who have survived breast cancer beyond 5 years without recurrence. A qualitative descriptive approach was used to collect and analyze the stories of 25 women, 40-78 years of age, with 5-26 years of survival time. Informants participated in three interviews that were transcribed and analyzed. Informants described "going through" a survival process that involved movement through several phases. The phases included interpreting the diagnosis, confronting mortality, reprioritizing, coming to terms, moving on, and flashing back. Phases were interpreted within the context of informants' backgrounds, sources of meaning, and explanatory models of understanding illness. Many informants described the emergence of a more authentic self as a result of the cancer experience. Many informants emerged from the cancer experience with a clearer sense of self, gratitude for life, and strength and confidence in their ability to manage life crises. Findings suggest that care might best be provided by understanding the context of each person's life.

Women's experiences of lymphedema.

PURPOSE/OBJECTIVES: To explore women's experiences of lymphedema. DESIGN: Qualitative descriptive. SETTING: An urban community in the midwestern United States. SAMPLE: Ten women who experienced lymphedema after breast cancer treatment and who had (a) completed their treatment for stage I or stage II breast cancer at least one year before the study, (b) experienced an onset of lymphedema at least two months after surgery, (c) no current evidence of cancer disease or cancer recurrence, (d) lymphedema not caused by cancer in the brachial plexus, and (e) no history of hospitalization for alcoholism, substance abuse, or psychiatric conditions. The women ranged in age from 36-75 years. Mean survival time was seven years, and the mean time since onset of lymphedema was four years. METHODS: Two in-depth interviews per participant. PATIENTS: Most women were able to continue living their normal lives. Some women experienced depression, anxiety, and impairments related to their intimate, work, and social relationships. Physicians' limited knowledge about lymphedema, conflicting treatment information, and the limited number of available treatment centers caused distress for the participants. Their experiences can be categorized into three predominant themes: Abandonment by Medicine, Concealing the imperfect image, and Living the Interrupted Life. CONCLUSIONS: Research efforts to expand the knowledge base regarding the epidemiology, prevention, and treatment of lymphedema are needed. Also needed is research that explores the impact of lymphedema on quality of life and functional ability as well as the psychosocial impact of lymphedema on body image, self esteem, and social support. IMPLICATIONS FOR NURSING PRACTICE: Care providers and breast cancer survivors should be educated about the prevention and treatment of lympedema. Nurses should refer patients to advocacy hot lines and support groups for information and support when appropriate. Women with lymphedema should be assessed periodically for psychosocial distress and referred for care as needed.

The presence of transforming growth factor e in bovine mammary gland.

Transforming growth factor e (TGFe) was demonstrated immunohistochemically in the bovine mammary gland, mainly in the glandular and ductal epithelium. In the teat, its expression was largely limited to the skin keratinocytes, ductal epithelium and ductal glands. It is suggested that this growth factor plays a role in lactation.

Human responses to simulated chemical warfare training in U.S. Army Reserve personnel.

This study examines biopsychological responses and background practices of 182 military personnel participating in a simulated chemical defense warfare scenario. The study explores the relationship between demographic, training, biopsychological response, and termination variables. Common features of dropouts are described.

A locking bar modification for splint localisation.

Currently used techniques for the construction of localising bars are generally regarded to constitute a disadvantage of cap splints. A simplified method of localisation is described.