Previously, we constructed a retrovirus vector (LCFSN) for transduction and expression of the cDNA encoding the normal human cystic fibrosis transmembrane conductance regulator (CFTR). The titer of virus from amphotropic packaging cells producing the LCFSN vector was low (10(3)-10(4) infectious units/ml). In an attempt to increase virus production, we used sodium butyrate (NaB) to treat murine retrovirus packaging cells producing this vector. NaB treatment increased the production of LCFSN from between 20-fold to greater than 1,000-fold, depending upon the producer clone, thereby resulting in virus titers up to about 1 x 10(7) infectious units/ml. This induction of virus titer could be accounted for, at least in part, by an increase in steady-state levels of full-length vector RNA within the producer cells. With some clonal producer cell lines, lowering the temperature of the virus harvest in combination with NaB treatment resulted in an apparent synergistic increase in virus production. The production of retrovirus vectors containing genes other than CFTR could also be increased by NaB treatment, although the enhancement in titer was modest (2-fold to 10-fold). The increase in virus production was not accompanied by an induction of replication-competent helper virus. NaB treatment also increased the transient production of retroviral vectors following DNA-mediated transfection into packaging cells such that virus titers of greater than 10(6) infectious units/ml could be readily attained.

Background A limitation to efficient lentivirus-mediated airway gene transfer is the lack of receptors to commonly used viral envelopes on the luminal surface of airway epithelia. The use of viral envelopes with natural tropism to the airway could be useful for overcoming this limitation. Methods We investigated influenza hemagglutinin (HA) pseudotyped EIAV-derived lentiviral vector-mediated gene transfer to the airway epithelium of adult and newborn mice. For these studies high-titer vectors were delivered by intranasal administration. In addition, we tested the feasibility of vector re-dosing to the nasal airway. Results Delivery of high-titer HA pseudotyped lentiviral vectors by nasal administration to newborn mouse pups or adult mice results in efficient transduction of airway epithelial cells in the nose, trachea, and lungs. In the nose vector expression was predominant in the respiratory epithelium and was not observed in the olfactory epithelium. In the trachea and large airways of the lung approximately 46% and 40%, respectively, of surface epithelial cells could be transduced. The efficiency of re-dosing to the nasal airway of mice was found to be dependent upon the age of the animal when the first dose is administered and the length of time between doses. Conclusions A single intranasal dose of concentrated influenza HA-pseudotyped lentiviral vector is sufficient for efficient gene transfer to the airways of mice. This is a promising result that could lead to the development of effective gene transfer reagents for the treatment of cystic fibrosis and other human lung diseases.

The sera of 21 different species of primates were surveyed for the presence of a trypanocidal factor to a monomorphic human serum-sensitive clone of Trypanosoma brucei gambiense (T.b.g.); human, gorilla, baboon (2 species), and the mandrill were found to contain this factor. The factor in all the sera is in the high density lipoprotein (HDL) fraction, and has similar modes of biological action. It has been shown that the human and gorilla trypanocidal factor share cross-reactive antigenic epitopes, but do not share similar cross-reactive epitopes with the baboon and mandrill factor. There was no relationship between the presence or absence of this factor and the primate's position on the phylogenetic tree. In addition, there was also no obvious correlation between the animals' preferred diet, and the presence or absence of trypanocidal activity. The evidence to date suggests that only African ground-dwelling primates that live in tsetse endemic areas contain the trypanocidal factor. It is assumed that this factor is involved in resistance of these primates to T.b.b. We believe that the host has developed trypanocidal substances as a result of selective evolutionary pressure by the African trypanosomes.

Primary ciliary dyskinesia is a genetically heterogeneous autosomal recessive disease in which mutations disrupt ciliary function, leading to impaired mucociliary clearance and life-long lung disease. Mouse tracheal cells with a targeted deletion in the axonemal dynein intermediated chain gene Dnaic1 differentiate normally in culture but lack ciliary activity. Gene transfer to undifferentiated cultures of mouse Dnaic1−/− cells with a lentiviral vector pseudotyped with avian influenza hemagglutinin restored Dnaic1 expression and ciliary activity. Importantly, apical treatment of well-differentiated cultures of mouse Dnaic1−/− with lentiviral vector also restored ciliary activity, demonstrating successful gene transfer from the apical surface. Treatment of Dnaic1flox/flox mice expressing an estrogen responsive Cre recombinase with different doses of tamoxifen indicated that restoration of ~20% of ciliary activity may be sufficient to prevent the development of rhinosinusitis. However, while administration of a β-galactosidase expressing vector to control mice demonstrated efficient gene transfer to the nasal epithelium, treatment of Dnaic1−/− mice resulted in a low level of gene transfer, demonstrating that the severe rhinitis present in these animals impedes gene transfer. The results demonstrate that gene replacement therapy may be a viable treatment option for primary ciliary dyskinesia, but further improvements in the efficiency of gene transfer are necessary.

The transformation of the long slender to the short stumpy stages of the African trypanosomes is an essential part of the trypanosome life cycle. Four possible mechanisms which could control this event have been investigated. It has been shown that (a) the dividing long slender to non-dividing short stumpy transition is not a programmed event in the trypanosome life cycle; nor (b) would it appear to be initiated by some form of cell to cell contact inhibition of growth. In addition, evidence is presented which would suggest that (c) the transition is not started by the depletion of a critical growth nutrient from the environment during the growth of the trypanosomes. The last possibility (d) considered is that during trypanosome growth, a growth inhibitor-short stumpy inducer accumulates in the trypanosomes' environment. Evidence is presented which shows that plasma from infected animals can inhibit the incorporation of thymidine by the trypanosomes. These data are consistent with the suggestion of an exogenous growth inhibitor accumulating during the infection.