Characterization of the oligosaccharide structures associated with the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane-associated glycoprotein. The protein can exist in three different molecular weight forms of approximately 127, 131, and 160 kDa, representing either nonglycosylated, core glycosylated, or fully mature, complex glycosylated CFTR, respectively. The most common mutation in cystic fibrosis (CF) results in the synthesis of a variant (DeltaF508-CFTR) that is incompletely glycosylated and defective in its trafficking to the cell surface. In this study, we have analyzed the oligosaccharide structures associated with the different forms of recombinant CFTR, by expressing and purifying the channel protein from either mammalian Chinese hamster ovary (CHO) or insect Sf9 cells. Using glycosidases and FACE analysis (fluorophore-assisted carbohydrate electrophoresis) we determined that purified CHO-CFTR contained polylactosaminoglycan (PL) sequences, while Sf9-CFTR had only oligomannosidic saccharides with fucosylation on the innermost GlcNAc. The presence of PL sequences on the recombinant CHO-CFTR is consistent with a normal feature of mammalian processing, since endogenous CFTR isolated from T84 cells displayed a similar pattern of glycosylation. The present study also reports on the use of FACE for the qualitative analysis of small amounts of glycoprotein oligosaccharides released enzymatically.

Targeting a recombinant adenovirus vector to HCC cells using a bifunctional Fab-antibody conjugate.

We developed a specific adenoviral gene delivery system with monoclonal antibody (mAb) AF-20 that binds to a 180 kDa antigen highly expressed on human hepatocellular carcinoma (HCC) cells. A bifunctional Fab-antibody conjugate (2Hx-2-AF-20) was generated through AF-20 mAb crosslinkage to an anti-hexon antibody Fab fragment. Uptake of adenoviral particles and gene expression was examined in FOCUS HCC and NIH 3T3 cells by immunofluorescence; beta-galactosidase expression levels were determined following competitive inhibition of adenoviral CAR receptor by excess fibre knob protein. The chimeric complex was rapidly internalized at 37 degrees C, and enhanced levels of reporter gene expression was observed in AF-20 antigen positive HCC cells, but not in AF-20 antigen negative NIH 3T3 control cells. Targeting of recombinant adenoviral vectors to a tumor associated antigen by a bifunctional Fab-antibody conjugate is a promising approach to enhance specificity and efficiency of gene delivery to HCC.

Scaleable chromatographic purification process for recombinant adeno-associated virus (rAAV).

BACKGROUND: Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Traditionally AAV has been purified from cell lysates using CsCl gradients; this approach however is not likely to be useful in large-scale manufacturing. Moreover gradient-purified AAV vectors tend to be contaminated with significant levels of cellular and adenoviral proteins and nucleic acid. To address the issue of purification we have developed a process scale method for the rapid and efficient purification of recombinant AAV (rAAV) from crude cellular lysates. METHODS: The preferred method for the purification of rAAVbetagal includes treatment of virally infected cell lysates with both trypsin and nuclease followed by ion exchange chromatography using ceramic hydroxyapatite and DEAE-Sepharose in combination with cellufine sulphate affinity chromatography. RESULTS: Purification of rAAV particles from crude cellular lysates co-infected with adenovirus was achieved using column chromatography exclusively. Column-purified rAAV was shown to be greater than 90% pure, free of any detectable contaminating adenovirus, biologically active, and capable of directing efficient gene transfer to the lungs of both cotton rats and mice. CONCLUSIONS: This study demonstrates the feasibility of using column chromatography alone for the isolation of highly purified rAAV vector. The methods described here are advancements in procedures to purify rAAV and are adaptable for commercial production of clinical-grade rAAV vector.

PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo.

Replication-defective recombinant adenovirus (Ad) vectors are under development for a wide variety of gene therapy indications. A potential limiting factor associated with virus gene therapy requiring repeated treatment is the development of a humoral immune response to the vector by the host. In animal models, there is a dose-dependent rise in neutralizing antibodies after primary vector administration, which can preclude effective repeat administration. The strategy we have developed to circumvent the neutralization of adenovirus vectors by antibodies is to mask their surface by covalent attachment of the polymer polyethylene glycol (PEG). Covalent attachment of PEG to the surface of the adenovirus was achieved primarily by using activated PEG tresylmonomethoxypolyethylene glycol (TMPEG), which reacts preferentially with the epsilon-amino terminal of lysine residues. We show that the components of the capsid that elicit a neutralizing immune response, i.e., hexon, fiber, and penton base, are also the main targets for PEGylation. Several protocols for PEGylation of an adenovirus vector were evaluated with respect to retention of virus infectivity and masking from antibody neutralization. We show that covalent attachment of polymer to the surface of the adenovirus can be achieved with retention of infectivity. We show further that PEG-modified adenovirus can be protected from antibody neutralization in the lungs of mice with high antibody titers to adenovirus, suggesting that PEGylation will improve the ability to administer Ad vectors on a repeated basis.

[Precocious neonatal mortality in premature newborns with low weight at birth and death]. / Mortalidad neonatal precoz en recien nacidos prematuros de bajo peso al nacer y de termino.

PIP: A retrospective study was conducted of all live births occurring in 1992 at a Dominican Institute of Social Security hospital in Santo Domingo to analyze the association between prematurity, low birth weight, and early neonatal mortality. Stillbirths, infants weighing less than 1000 g or born before 28 weeks of gestation, and those with lethal malformations were excluded. 5142 newborns met the inclusion criteria. 1701 deliveries (33%) were cesarean. 550 of the newborns (10.7%) were low birth weight, and 338 (6.6%) were premature. The early neonatal mortality rate was 17/1000. Low birth weight infants accounted for 66.2% of early neonatal mortality. 10.7% of low birth weight infants died within the first week of life, and their relative risk of early neonatal death was 16.42. 64% of all infants dying in their first week of life were also premature. The specific mortality rate for premature infants was 168.6/1000 live births. The relative risk was 25.32 for premature infants. Low birth weight infants born at term had an early neonatal mortality rate of 24.6/1000 live births, compared to 5.5/1000 for term births of adequate weight.^ieng