A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer.

Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of beta-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases beta-galactosidase expression 90-fold in SMC in vitro and approximately 40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies.

Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination.

Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus. By using a very efficient recombinase, in vitro-evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography-based virus purification.

Gene transfer into rat brain using adenoviral vectors.

Recombinant adenovirus vectors are attractive vehicles to deliver genes into the brain for the purposes of neurobiological research and for gene therapy of neurological diseases. This unit provides a comprehensive set of protocols for adenovirus vector-mediated gene transfer to the brain, including introduction of the vector into the brain by stereotaxic injection and preparation and processing of brain tissue for the evaluation of gene transfer. The potential side-effects of administering adenovirus vectors to the brain are discussed in detail. The unit also provides protocols for evaluating these side-effects (e.g., demyelination, inflammation, vector-mediated cytotoxicity, etc.). Finally, critical parameters for obtaining optimal gene transfer with minimum side-effects are presented.

Strong promoters are the key to highly efficient, noninflammatory and noncytotoxic adenoviral-mediated transgene delivery into the brain in vivo.

Using the major immediate early murine cytomegalovirus (MIEmCMV) promoter to drive expression of beta-galactosidase, we have demonstrated that, following adenoviral-mediated transduction of brain cells in vivo, a single viral infectious unit is capable of producing detectable levels of transgene expression and that gene transfer into the brain is close to 100% efficient. By reducing 100-fold the amount of virus needed to detect large numbers of transduced brain cells, we were able to completely eliminate the cellular inflammation and viral cytotoxicity associated with the delivery of adenoviral vectors into the brain compared to saline-injected controls. These results demonstrate that a strong promoter is necessary to allow the use of low concentrations of adenoviral vectors for gene transfer into the brain, thereby eliminating deleterious side effects and increasing the potential efficacy of gene therapy.

Transcriptional targeting to anterior pituitary lactotrophic cells using recombinant adenovirus vectors in vitro and in vivo in normal and estrogen/sulpiride-induced hyperplastic anterior pituitaries.

The use of pituitary cell type-specific promoters is a powerful molecular tool to achieve pituitary cell type-specific transcriptional targeting of transgenes encoded by viral vectors. It has recently been proposed that transcriptional targeting of therapeutic genes could be harnessed as a gene therapy strategy for the treatment of pituitary disease. We describe the successful use of the human PRL promoter (hPrl) encoded within recombinant adenovirus vectors to target transgene expression of Herpes Simplex Virus Type 1-Thymidine Kinase (HSV1-TK) or beta-galactosidase to lactotrophic cells in vitro and in vivo. Functionally, the restriction of expression of HSV1-TK to lactotrophic tumor cells, using the hPrl promoter, resulted in the cell type-specific induction of apoptosis in the lactotrophic GH3 tumor cell line, in the presence of ganciclovir (GCV). In the corticotrophic AtT20 cell line, we detected neither HSV1-TK expression, nor apoptosis in the presence of GCV. The hPrl promoter encoded within a recombinant adenoviral vector also restricted transgene expression to lactotrophic cells in primary anterior pituitary (AP) cultures, and importantly, within the anterior pituitary gland in vivo. When the HSV1-TK driven by hPrl promoter was used in an in vivo model ofestrogen/sulpiride lactotroph induced hyperplasia within the AP in situ, the treatment was not effective in either reducing the weight of the gland, the number of lactotrophic cells within the transduced area in vivo, or the circulating PRL levels. This is in contrast to the human cytomegalovirus promoter (hCMV) driving expression of HSV1-TK in the same experimental paradigm, which was effective in reducing pituitary weight and circulating PRL levels. Our results have important implications in the design of gene therapy strategies for pituitary tumors. We demonstrate that both the choice of the in vivo animal model, i.e. adenoma in the AP gland in situ, and the particular gene therapy strategy chosen, i.e. use of strong ubiquitous promoters vs. weaker but cell type-specific promoters, determine the experimental therapeutic outcome.

'Complete' spinal cord injury does not block perceptual responses to genital self-stimulation in women.

BACKGROUND: A priori hypothesis: vaginal and/or cervical self-stimulation will not produce perceptual responses in women with "complete" spinal cord injury (SCI) at or above the highest level of entry of the hypogastric nerves (T10-12) but will produce perceptual responses if SCI is below T-10. DESIGN: Women with complete SCI were assigned to a group with "upper" (T-10 and/or above) (n = 6) or "lower" (below T-10) (n = 10) SCI; uninjured women (n = 5) constituted a control group. Perceptual response to vaginal and/or cervical self-stimulation was quantified as magnitude of analgesia to calibrated finger compressive force. SETTING: Rutgers, The State University of New Jersey, Human Physiology Laboratory, College of Nursing, Newark. PARTICIPANTS: Consecutive samples of first 16 of 34 women with SCI who responded to nationwide advertisements, met inclusion criteria, and volunteered; control group was the first 5 respondents. INTERVENTION: Vaginal or cervical (cervix uteri) self-stimulation applied for 12 minutes, interspersed with non-stimulation periods, while measuring analgesia. MAIN OUTCOME MEASURE: Quantify analgesia magnitude to vaginal or cervical self-stimulation. RESULTS: Significant analgesia was produced in the uninjured group and the group with lower SCI, supporting the hypothesis. Unexpectedly, significant analgesia was also produced in the group with upper SCI. Women in the group with upper SCI also experienced menstrual discomfort, awareness of vaginal and/or cervical stimulation per se, and orgasms. CONCLUSIONS: (1) Genitospinal visceral afferent pathways function in the women in the group with upper SCI, although unrecognized by the American Spinal Injury Association criteria, and/or (2) there exists a functional genital afferent pathway that bypasses the spinal cord and projects directly to the brain, which we propose to be via the vagus nerves.

Fts insertional mutant of Salmonella typhimurium.

A temperature-sensitive filamentation (fts) Salmonella typhimurium mutant was isolated after transposon mutagenesis with mini-Tn 10dTc. The mutant was unable to form colonies after 20 h incubation at 37 degrees C on LB agar. Colonies appeared, however, after longer incubation at the restrictive temperature. Filamentation affected only part of the bacterial population. Rapid mapping using Mu dP22 hybrid phages revealed that the mutation, ftsD220, lies within minutes 68.5 and 73.6 on the genetic map. Further analysis revealed that the ftsD220 mapped at min 73 and that it is linked to cysG (6%) and to aroB (39%). Complementation tests suggested that the ftsD220 mutation is not homologous to a Escherichia coli ftsH mutation.