Virus-mediated Transgene Expression Research Articles

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Hoechst increases adeno‐associated virus‐mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter

In airway epithelia, the kinetics of recombinant adeno-associated virus (AAV) transgene expression is slow. This has negative practical implications for research, as well as for translation into therapy. The DNA minor groove-binding agent Hoechst-33342 has been shown to enhance AAV transgene expression. In the present study, we investigated the mechanism of Hoechst-related augmentation of AAV-mediated transgene expression. We investigated the effect of Hoechst-33342 on HT1080, COS-7, mouse and human airway epithelia transduced with different AAV serotypes encoding enhanced green fluorescent protein (eGFP). We exposed cells to increasing concentrations of Hoechst-33342 at different time points. We evaluated the effect on second-strand DNA synthesis using AAV with a self-complementary genome. We also investigated the effect on expression from transfected plasmids with and without AAV2 inverted terminal repeats (ITRs). We found that Hoechst-33342 significantly accelerated AAV transgene expression for all serotypes tested. Hoechst-33342 only had an effect when the treatment was given during or after transduction, even 120 days post-transduction, suggesting an effect on transgene expression regulation. Hoechst-33342 increased transgene expression when cells were transduced with a self-complementary AAV with the cytomegalovirus promoter, although there was no effect on cells transduced with conventional single-stranded AAV encoding the Rous sarcoma virus promoter. Finally, Hoechst-33342 increases gene expression from transfected plasmids regardless of the presence of AAV2 ITRs. Hoechst dramatically augments and accelerates AAV-mediated transgene expression in airway epithelia without altering AAV-mediated gene transfer. Hoechst activation of the cytomegalovirus promoter is seen in plasmids, although it is drastically enhanced in the context of AAV.

In vivo and in vitro effects of ultrasound or/and microbubbles on recombinant adeno-associated virus-mediated transgene expression in the retina

Background: Ultrasound-targeted microbubble destruction has been utilized to deliver a drug/gene into cells in vitro and in vivo. Objective: To access the effects of ultrasound or/and microbubbles on recombinant adeno-associated virus (rAAV)-mediated transgene expression in rat retinal pigmented epithelium-J (RPE-J) cells in vitro and Wistar rat retina in vivo and to compare the difference between them. Methods: Different doses of serotype-2 rAAV (rAAV2) encoding an enhanced green fluorescent protein gene and microbubbles were administered to RPE-J cells in vitro and Wistar rat retina in vivo under different ultrasound conditions. Transfection efficiency was assessed by fluorescence microscopy, fluorescence stereoscope and flow cytometry analysis. Cell viability and tissue damage were assessed by trypan blue staining and hematoxylineosin staining. Results: rAAV2-mediated transgene expression in vivo was significantly enhanced by ultrasound and microbubbles, while rAAV2-mediated gene transfection in vitro was significantly enhanced by ultrasound or microbubbles alone. Conclusion: Ultrasound or/and microbubbles-mediated rAAV delivery strategies must be chosen for retinal gene therapy based on their effects in vitro and in vivo.

118. The Histone Deacetylase Inhibitor Enhanced Recombinant Adeno-Associated Virus-Mediated Transgene Expression in Cancer Cells

118. The Histone Deacetylase Inhibitor Enhanced Recombinant Adeno-Associated Virus-Mediated Transgene Expression in Cancer Cells

Adenovirus-mediated gene transfer to renal glomeruli in rodents

The expression of foreign genes into renal glomerular cells holds enormous potential to modulate the outcome of renal diseases. Recombinant adenoviruses (rAds) are promising gene transfer vectors because they have the ability to infect a wide range of nondividing cells. However, despite the fact that renal glomeruli are easily accessible via the renal circulation, adenovirus-mediated gene transfer into rodent glomeruli has been problematic. Here, we described our experience using rAd vectors to express foreign genes in rodent renal glomeruli in vivo and in cultured human renal glomerular cells. We developed two techniques--the "portal clamping" and "prolonged renal infusion"--to infect mouse and rat renal glomeruli in vivo, respectively. We used E-1-deleted rAd vectors carrying the lacZ gene encoding beta-galactosidase (Ad. CBlacZ) under the control of the cytomegalovirus enhancer and chicken beta-actin promoter. Cultured human renal glomerular podocytes, endothelial and mesangial cells were grown following standard techniques. Transgene expression was evaluated by doing beta-galactosidase staining and reverse transcription-polymerase chain reaction studies. We found that both a prolonged exposure and a high concentration of circulating adenoviral vectors were required to achieve efficient gene transfer to renal glomerular cells in rodents. The virus-mediated transgene expression in renal glomeruli lasted for at least 42 days in mice and 21 days in rats without causing significant renal injury. These data demonstrate the feasibility of using rAd vectors as a tool to express foreign genes in rodent renal glomerular cells and suggest that all types of human renal glomerular cells are equally susceptible to rAd infection.

Perfluorochemical liquid enhances adeno-associated virus-mediated transgene expression in lungs.

Use of adeno-associated virus (AAV) vectors for lung gene therapy is limited, in part, by low levels of AAV-mediated transgene expression in lungs. Generally, less than 1% of total airway and alveolar epithelial cells express transgene activity following vector administration. A means of improving AAV vector delivery could potentially enhance AAV-mediated gene expression in lungs. We have previously demonstrated that use of perfluorochemical (PFC) liquids improved overall levels of adenovirus vector-mediated gene expression as well as distribution of expression in lungs of spontaneously breathing rodents. To evaluate whether use of PFC liquids might similarly enhance AAV-mediated expression, spontaneously breathing rodents received intratracheal instillation of the AAV vectors CWRAP and ARAP4 (2-5 x 10(8) FFU/animal) with or without 10 cc/kg body wt PFC liquid (FC-75, ACROS). Animals were sacrificed 4 weeks later and lungs assessed for overall and in situ alkaline phosphatase (AP) expression. Animals receiving vector alone exhibited scattered sparse in situ activity, predominantly in alveolar epithelium. In contrast, animals receiving vector with FC-75 exhibited increased and more widespread AP expression as well as up to a 26-fold increase in AP activity. These results demonstrate that use of the PFC liquid FC-75 improves overall and in situ AAV-mediated gene expression in rodent lungs.

Transduction of the contralateral ear after adenovirus-mediated cochlear gene transfer.

Cochlear gene transfer is a promising new approach for inner ear therapy. Previous studies have demonstrated hair cell protection with cochlear gene transfer not only in the inoculated, but also in the uninoculated ear. To characterize the kinetics of viral spread, we investigated the extent of transgene expression in the contralateral (uninoculated) cochlea after unilateral adenoviral cochlear gene transfer. We used a lacZ reporter gene vector, and demonstrated spread of the adenovirus into the cerebrospinal fluid (CSF) after cochlear inoculation of 25 microl viral vector. Direct virus application into the CSF resulted in transduction of both cochleae, whereas virus inoculation into the bloodstream did not. The cochlear aqueduct was identified as the most likely route of virus spread to the contralateral cochlea. These data enhance our understanding of the kinetics of virus-mediated transgene expression in the inner ear, and assist in the development of clinical applications for inner ear gene therapy. Our results showed a functional communication between the CSF and the perilymphatic space of the inner ear, that is not only of importance for otological gene transfer, but also for CNS gene transfer. Gene Therapy (2000) 7, 377-383.