Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors

Plasma apolipoprotein AI (apoAI) and lecithin-cholesterol acyltransferase (LCAT) play important roles in reverse cholesterol transport, promoting the removal of excess cholesterol from peripheral cells and reducing formation of atherosclerotic lesions. Gene augmentation of either apoAI or LCAT, or both, are thus attractive targets for prevention or treatment of atherosclerosis. With the eventual aim of safe and efficient gene delivery to skeletal muscle, our chosen secretory platform for systemic delivery of anti-atherogenic proteins, we have constructed conventional and AAV-based plasmid vectors containing human apoAI or LCAT cDNAs; their efficacy was tested by lipoplex transfection of mouse C2C12 muscle cells or human 293 cells. The secretion of apoAI or LCAT by transduced cultures was two- to five-fold higher using AAV-based plasmid vectors than conventional plasmid vectors. Additionally, cells transfected with a bicistronic AAV-based vector containing an internal ribosome entry site (IRES) efficiently expressed both apoAI and LCAT simultaneously. Furthermore, AAV-based vector sequences were retained by host cells, whereas those of conventional plasmid vectors were lost. These studies indicate that ectopic overexpression of apoAI and LCAT in muscle tissue using AAV-based plasmid vectors might provide a feasible anti-atherogenic strategy in vivo.     

Antagonistic effect of human alpha-1-antitrypsin on excystation of Cryptosporidium parvum oocysts

This study evaluated the effects of the human serine protease inhibitor alpha-1-antitrypsin (AAT) on in vitro excystation and infectivity of Cryptosporidium parvum. Excystation was monitored at 37 C in RPMI medium in the presence of 0, 100, 500, or 1,000 micrograms/ml AAT. AAT significantly inhibited (P < 0.05) excystation of bleach-decontaminated oocysts in a concentration-dependent manner at incubation intervals from 15 to 90 min but did not alter the excystation dynamics of unbleached oocysts. Bleach-treated oocysts, suspended in RPMI containing 0, 1, 10, 100, 500, or 1,000 micrograms/ml AAT, were used to inoculate bovine fallopian tube epithelial (BFTE) cell monolayers. Alternately, sporozoites, excysted at 37 degrees C and collected by filtration, were used to inoculate BFTE cells under the same conditions. The mean number of parasites counted in AAT-treated, oocyst-inoculated cells was significantly less (P < 0.01) than control mean values at 24 and 48 hr post-inoculation (PI); longer PI intervals (72-96 hr) exhibited a decreased inhibitory effect. AAT did not inhibit parasite infection when cultures were inoculated with C. parvum sporozoites. The findings of this study show that the anticryptosporidial potential of AAT is primarily associated with an antagonistic effect on oocyst excystation.     

Alleles of the alpha-1-antitrypsin phenotype in patients with aortic aneurysms

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurodevelopmental disorders with interrelated genetic mechanisms because genomic imprinting within the chromosome 15q11-13 region affects both the PWS and the AS locus. Methylation analysis is one method of distinguishing between the maternally and paternally inherited chromosome 15. Here we present clinical and molecular data on a large series of 258 referred patients, evaluated with methylation analysis: 115 with suspected PWS and 143 with suspected AS. In these patients, the clinical phenotype was graded into three groups: classical (group 1); not classical but possible (group 2); not classical and unlikely (group 3). For PWS, a fourth group consisted of hypotonic babies. DNA methylation analysis confirmed the diagnosis of PWS in 30 patients (26%) and AS in 28 patients (20%). For 21 PWS patients the mechanism was established: 15 had deletions, 4 had uniparental disomy (UPD) and 2 a presumed imprinting defect. Clinically all those with an abnormal methylation pattern had the classical phenotype and none of those with a normal methylation pattern had classical PWS. For 23 AS patients in whom a mechanism was established, 17 had a deletion, 3 had UPD and 3 had a presumed imprinting defect. There was greater clinical overlap in AS, with 26 classical AS patients having a normal methylation pattern while an abnormal methylation pattern was seen in one patient from group 2. In addition, there were a further 40 patients with a normal methylation pattern in whom AS was still a possible diagnosis. Our conclusion is that methylation analysis provides an excellent screening test for both syndromes, providing approximately 99% diagnosis for PWS and for AS, a 75% diagnostic rate, supplemented for the remaining 25% with an essential basic starting point to further investigations.     

Genetic immunization with adeno-associated virus vectors expressing herpes simplex virus type 2 glycoproteins B and D

Intramuscular injection of mice with an adeno-associated virus (AAV) vector expressing herpes simplex virus type 2 glycoprotein B led to the generation of both gB-specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes and anti-gB antibody. AAV-mediated immunization was more potent than plasmid DNA or protein in generating antibody responses.     

Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes

Adeno-associated virus (AAV)-based vectors have been shown to be effective in transferring the cystic fibrosis gene (CFTR) into airway epithelial cells in animal models and in patients. However, the level of CFTR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. In this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporation of an effective promoter with the CFTR gene into AAV vectors. We engineered and tested 20 CFTR mini-genes containing deletions that were targeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs (one with combined deletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and regulation by cAMP. By using an AAV vector with a P5 promoter, we transduced these short forms of CFTR genes into target cells and demonstrated high levels of CFTR expression. We also demonstrated that smaller AAV/CFTR vectors with a P5 promoter expressed the CFTR gene more efficiently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene with combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively transferred CFTR function into target cells. These new vectors should circumvent the limitations of AAV vector for CFTR expression. Our strategy also may be applicable to other genes, the sizes of which exceed the packaging limit of an AAV vector.     

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.     

Optimization of packaging of adeno-associated virus gene therapy vectors using plasmid transfections

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC-ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100x1 mm I.D. C8, 5 microm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 microl/min. For monoamines and metabolites we used a 150X1 mm I.D. C18 5 microm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 microM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-microm tissue slices. Tissue samples were homogenized in 50 or 100 microl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and gamma-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

Should we administer replacement therapy to patients with alpha-1-antitrypsin deficit?

Comments are given on the present recommendations for the keeping of horses in stables. Proposals for an animal friendly accommodation are made including practical considerations.     

Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus

Recently, efficient and long-term in vivo gene transfer by recombinant adeno-associated virus type 2 (rAAV) vectors has been demonstrated in a variety of tissues. Further improvement in vector titer and purity will expedite this in vivo exploration and provide preclinical information required for use in human gene therapy. In an effort to obtain higher titers, we constructed a novel AAV helper plasmid which utilizes translational control of AAV Rep genes (J. Li et al., J. Virol. 71:5236-5243, 1997). To address the issue of purity, in this study we report the first rAAV production method which is completely free of adenovirus (Ad) helper virus. The new production system uses a plasmid construct which contains a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes. This construct is missing some of the early and most of the late Ad genes and is incapable of producing infectious Ad. Transfection of 293 cells with the new mini-Ad helper and AAV packaging plasmids results in high-titer rAAV vectors with yields greater than 1,000 transducing units, or 10(5) viral particles per cell. When rAAV vectors were produced by using this production scheme and compared to traditional heat-inactivated rAAV preparations in vitro and in vivo, we observed transduction equivalent to or better than normal levels. The complete removal of infectious Ad from AAV production should facilitate a better understanding of immune response to AAV vectors in vivo, eliminate the need for developing replication-competent Ad assays, and provide a more defined reagent for clinical use.     

Usefulness of the quantification of the alpha-1 serous protein band in the screening of alpha-1-antitrypsin deficiency

Dramatic aesthetic results have been obtained with the flexible silicone gingival mask which can be used to correct deformities remaining after destructive periodontal inflammation has been controlled. The silicone mask may also be used as an interim measure to improve the appearance of anterior crowns after initial periodontal therapy to allow time for healing and the establishment of periodontal stability and prognosis. A simple two-stage impression technique is described, enabling a suitably trained dental technician to produce comfortable and accurately fitting masks, which are very stable during use. Virtually no problems have been encountered.     

Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2

Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.     

Fibromuscular dysplasia of the internal carotid artery in a child with alpha-1-antitrypsin deficiency

Fibromuscular dysplasia (FMD) is a non-inflammatory segmental arteriopathy of unknown origin. Most often the renal arteries are affected, however, also mesenteric, lumbar, vertebral, or carotid arteries may be involved. FMD has frequently been reported as a cause of stroke in adults, but very rarely in children. We report the case of an 11-year-old boy who presented with an ischaemic infarction in the anterior part of the territory of the left middle cerebral artery. Angiography demonstrated a 'string of beads' lesion suggestive of FMD causing occlusion at the origin of the middle artery. Laboratory analyses revealed the protease inhibitor (Pi) phenotype SZ (PiSZ) of alpha-1-antitrypsin deficiency as well as decreased antioxidants and signs of enhanced lipid peroxidation. Such an imbalance may be associated with diminished resistance to oxidation, possibly causing direct cellular and tissue injury. Whether alpha-1-antitrypsin deficiency and an impaired status of antioxidants, as seen in our patient, might play a role in the pathogenesis of FMD is presently unclear.     

Multiplication of adeno-associated virus type 1 in cells coinfected with a temperature-sensitive mutant of human adenovirus type 31

The maturation of the stato-acoustic nerve in the cat was studied by light and electron microscopy from the fetal stage to the adult. Measurement of the outer diameter of the fibers and the study of the myelination process revealed that myelination begins earlier for the vestibular nerve than for the cochlear nerve: by the fifty-third day of gestation 64% of the vestibular fibres have already passed the promyelin stage whereas for the cochlear nerve this promyelin stage begins for the majority of fibers on the fifty-seventh gestation day. Afterward, maturation proceeds more rapidly for the cochlear nerve. In the case of both nerves, maturation is still incomplete at two months of age. Concerning the relationship between the thickness of the myelin sheath and the axoplasmic diameter, there is already a good correlation by the fifty-seventh day of gestation in the vestibular nerve, whereas it appears several days after birth in the cochlear nerve.     

Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle

The skeletal muscle provides a very permissive physiological environment for adeno-associated virus (AAV) type 2-mediated gene transfer. We have studied the early steps leading to the establishment of permanent transgene expression, after injection of recombinant AAV (rAAV) particles in the quadriceps muscle of mice. The animals received an rAAV encoding a secreted protein, murine erythropoietin (mEpo), under the control of the human cytomegalovirus major immediate-early promoter and were sacrificed between 1 and 60 days after injection. The measurement of plasma Epo levels and of hematocrits indicated a progressive increase of transgene expression over the first 2 weeks, followed by a stabilization at maximal plateau values. The rAAV sequences were analyzed by Southern blotting following neutral or alkaline gel electrophoresis of total DNA from injected muscles. While a high number of rAAV sequences were detected during the first 5 days following the injection, only a few percent of these sequences was retained in the animals analyzed after 2 weeks, in which transgene expression was maximal. Double-stranded DNA molecules resulting from de novo second-strand synthesis were detected as early as day 1, indicating that this crucial step of AAV-mediated gene transfer is readily accomplished in the muscle. The templates driving stable gene expression at later time points are low in copy number and structured as high-molecular-weight concatemers or interlocked circles. The presence of the circular form of the rAAV genomes at early time points suggests that the molecular transformations involved in the formation of stable concatemers may involve a rolling-circle type of DNA replication.     

Cervical human immunodeficiency virus type 1 shedding is associated with genital beta-chemokine secretion

Forty human immunodeficiency virus type 1 (HIV-1)-infected women participated in a cross-sectional study of possible correlations between chemokine receptor (CCR5 and/or CCR2B) genotype, HIV-1 RNA and DNA load, and beta-chemokine levels (RANTES, MIP-1alpha, MIP-1beta) in blood and cervix. HIV-1 nucleic acid and beta-chemokines were found in all patient blood samples and in more than half of the cervical samples regardless of CCR5 or CCR2B genotype. High beta-chemokine concentrations were in general associated with high virus loads in blood and cervix. In the blood, the proviral DNA load was significantly correlated with the MIP-1alpha concentration, whereas the DNA load in cervix was significantly associated with the MIP-1beta concentration. The cervical viral RNA load was significantly associated with levels of all three chemokines. Thus, when HIV-1 shedding was highest in the genital tract, it was associated with other combinations of beta-chemokines than virus load in blood, suggesting that local immune reactions strongly influence virus load in the cervical compartment.     

Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells

Lentiviruses are potentially advantageous compared to oncoretroviruses as gene transfer agents because they can infect nondividing cells. We demonstrate here that human immunodeficiency virus type 1 (HIV-1)-based vectors were highly efficient in transducing purified human hematopoietic stem cells. Transduction rates, measured by marker gene expression or by PCR of the integrated provirus, exceeded 50%, and transduction appeared to be independent of mitosis. Derivatives of HIV-1 were constructed to optimize the vector, and a deletion of most of Vif and Vpr was required to ensure the long-term persistence of transduced cells with relatively stable expression of the marker gene product. These results extend the utility of this lentivirus vector system.     

Usefulness of a national registry of alpha-1-antitrypsin deficiency. The Spanish experience

Severe alpha-1-antitrypsin (AAT) deficiency, phenotype Pi ZZ, is a rare condition with an estimated prevalence of 1/4500 individuals in Spain. Given this low prevalence, it seems useful to accumulate all the information derived from the care of these patients. In this context, the Spanish Registry of patients with AAT deficiency was founded in 1993; its main objectives were to establish guidelines adapted to our country for the treatment and management of AAT-deficient patients, offer expert support to physicians all over the country treating these patients, and provide technical support on the determination of Pi phenotyping and genotyping of individuals suspected of being AAT-deficient. From 1993 to January 1998 the number of enrollees increased from 48 to 223, of which 216 were Pi ZZ. Seventy-three per cent were male and only 31.5% were never smokers, mean age was 46 years (SD = 13 years) and mean FEV1 53% predicted (SD = 31%). 83% were index cases who, compared with non-index cases, were older (49 +/- 11 vs. 35 +/- 13 years, P < 0.001), more likely to have a smoking history (85% vs. 47%, P < 0.01) and displayed more severe impairment in pulmonary function (FEV1% = 40% +/- 19% vs. 96% +/- 23%, P < 0.001). Augmentation therapy was administered to 129 patients (58%). Treated patients had more severe impairment in pulmonary function than the untreated (FEV1% = 40% +/- 21% vs. 72% +/- 32%, P < 0.001) and were more likely to be index cases (81% vs. 43%, P < 0.001). Characteristics of the patients included are similar to those described for other Registries. The Registry has extended knowledge of the disease throughout the country and has established local guidelines for treatment and follow-up. It may be a valid database for future co-operation in international initiatives.     

Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells

We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication.     

Low density lipoprotein catabolism is enhanced by the cleaved form of alpha-1-antitrypsin

The frequent occurrence of hypocholesterolaemia following inflammatory processes is well known but unexplained. Elevated plasma levels of serine proteinase inhibitors (serpins) and their complexes with target enzymes have been demonstrated in inflammatory, malignant and infectious diseases which are also often accompanied by low plasma cholesterol levels. Under inflammatory conditions, uncomplexed, but cleaved inactive serpins arising from slow deacylation of the serpin-proteinase complex may be present in the circulation. To determine the influence of native and cleaved forms of serpins, such as alpha-1-antitrypsin (AAT), on lipoprotein metabolism, we investigated the effect of these forms on low density lipoprotein (LDL) catabolism in human HepG2 cell line. We have found that the cleaved form of AAT in concentrations from 125 to 2000 nmol l-1 stimulates LDL binding to the HepG2 cells, by up to 49% with a subsequent increase in LDL uptake and degradation of up to 79 and 65% respectively. Native AAT was also found to increase LDL binding and internalization by 20-25%, independently of the amount of AAT added, an effect most probably due to the cleaved form of AAT produced by local proteolysis of native AAT incubated in the cell culture. Moreover we have shown that the cleaved form of AAT interacts with LDL in vitro, and that such an interaction abolishes AAT ability to stimulate LDL binding and internalization. This study for the first time describes the ability of the cleaved form of AAT to stimulate LDL binding and internalization in HepG2 cell culture, and provides evidence that hypocholesterolaemia occurring during inflammatory processes may be mediated by cleaved forms of serpins.     

Long-term therapy of alpha 1-antitrypsin-deficiency-associated pulmonary emphysema with human alpha 1-antitrypsin

alpha 1-antitrypsin (alpha 1-AT) deficiency is a genetic disorder characterized by low serum levels of alpha 1-AT and a high risk of pulmonary emphysema at a young age. The resulting surplus of proteases, mainly of neutrophil elastase, can be balanced by i.v. augmentation with alpha 1-AT. However, it is not clear if affected patients benefit from long-term augmentation therapy and no long-term safety data are available. We examined 443 patients with severe alpha 1-AT deficiency and pulmonary emphysema receiving weekly i.v. infusions of 60 mg/kg body weight alpha 1-AT in addition to their regular medication. The progression of the disease was assessed by repeated lung function measurements, particularly the decline in forced expiratory volume in 1 second (delta FEV1). 443 patients with alpha 1-AT deficiency tolerated augmentation therapy well with few adverse reactions. The delta FEV1 in 287 patients with available follow-up data was 57.1 +/- 31.1 ml per year. Stratified for baseline FEV1, the decline was 35.6 +/- 21.3 ml in the 108 patients with an initial FEV1 < 30% and 64.0 +/- 26.4 ml in the 164 with 30% < FEV1 < or = 65% of predicted normal (p = 0.0008). The remaining 15 patients had an initial FEV1 > 65%. Long-term treatment with i.v. alpha 1-antitrypsin in patients with severe alpha 1-Pi deficiency is feasible and safe. The decline in forced expiratory volume in one second is related to the initial forced expiratory volume in one second as in alpha 1-antitrypsin deficient patients not receiving augmentation therapy.