Vivo Gene Research Articles

827. Development of Novel AAV Vectors for Potential In Vivo Gene transfer to Hematopoietic Cells in Bone Marrow

827. Development of Novel AAV Vectors for Potential In Vivo Gene transfer to Hematopoietic Cells in Bone Marrow

677. In Vitro & Vivo Gene Targeting with Adenoviral Vector Conjugated Magnetic Nanobeads for Cardiac Regeneration

677. In Vitro & Vivo Gene Targeting with Adenoviral Vector Conjugated Magnetic Nanobeads for Cardiac Regeneration

Identification of MicroRNA Target Genes in Vivo

MicroRNAs (miRNAs) are short non-coding RNAs transcribed from intergenic or intronic sequences as long precursors that are sequentially processed by the endonucleases Drosha and Dicer into short double-stranded sequences. It is clear that miRNAs play essential roles in gene expression, development, and cell fate specification in animals. However, one of the barriers of miRNA research is how to find the target genes. In this study, we have developed a rapid and effective method to isolate miRNA target genes in vivo. MicroRNA was synthesized in vitro and labeled by biotin. After transfected into cells, the miRNA/mRNA complexes were isolated by streptavidin-coated magnetic beads. hsa-miR155 was taken as model to validate this method, which is a very important modulator in tumor development. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.

Preclinical and clinical progress in hemophilia gene therapy

Hemophilia A and B are attractive target diseases for gene therapy, as stable expression of coagulation factor VIII and IX may correct the bleeding diathesis. This review focuses on the recent progress in preclinical and clinical studies in gene therapy for hemophilia A and B. Hepatic gene delivery using vectors derived from adeno-associated virus (AAV) resulted in therapeutic but transient functional clotting factor IX (FIX) expression levels in severe hemophilia B patients. Although T-cell-mediated immune responses eliminated the transduced hepatocytes, transient immunosuppression may potentially overcome this limitation. Alternatively, vectors are being developed that result in higher FIX expression levels at lower vector doses. Lentiviral vectors are being explored for in-vivo hepatic gene delivery and for ex-vivo transduction of hematopoietic stem cells. This resulted in stable correction of the bleeding diathesis in hemophilic mice. Finally, nonviral vectors derived from transposons result in sustained clotting-factor expression in rodent models. Translational studies in large animal models are required to move these new approaches forward into the clinic. New insights from clinical trials and advances in preclinical studies may ultimately pave the way toward a cure in patients suffering from hemophilia.

Development of an ultrasound-responsive and mannose-modified gene carrier for DNA vaccine therapy

Development of a gene delivery system to transfer the gene of interest selectively and efficiently into targeted cells is essential for achievement of sufficient therapeutic effects by gene therapy. Here, we succeeded in developing the gene transfection method using ultrasound (US)-responsive and mannose-modified gene carriers, named Man-PEG 2000 bubble lipoplexes. Compared with the conventional lipofection method using mannose-modified carriers, this transfection method using Man-PEG 2000 bubble lipoplexes and US exposure enabled approximately 500∼800-fold higher gene expressions in the antigen presenting cells (APCs) selectively in vivo. This enhanced gene expression was contributed by the improvement of delivering efficiency of nucleic acids to the targeted organs, and by the increase of introducing efficiency of nucleic acids into the cytoplasm followed by US exposure. Moreover, high anti-tumor effects were demonstrated by applying this method to DNA vaccine therapy using ovalbumin (OVA)-expressing plasmid DNA (pDNA). This US-responsive and cell-specific gene delivery system can be widely applied to medical treatments such as vaccine therapy and anti-inflammation therapy, which its targeted cells are APCs, and our findings may help in establishing innovative methods for in-vivo gene delivery to overcome the poor introducing efficiency of carriers into cytoplasm which the major obstacle associated with gene delivery by non-viral carriers.

107. In Vivo Gene Delivery to the Skin by Electroporation Using a Multiple Electrode Array

107. In Vivo Gene Delivery to the Skin by Electroporation Using a Multiple Electrode Array

121. In Vivo Gene Delivery from Modified PLG Scaffolds

121. In Vivo Gene Delivery from Modified PLG Scaffolds

Gene therapy used for tissue engineering applications

This review highlights the advances at the interface between tissue engineering and gene therapy. There are a large number of reports on gene therapy in tissue engineering, and these cover a huge range of different engineered tissues, different vectors, scaffolds and methodology. The review considers separately in-vitro and in-vivo gene transfer methods. The in-vivo gene transfer method is described first, using either viral or non-viral vectors to repair various tissues with and without the use of scaffolds. The use of a scaffold can overcome some of the challenges associated with delivery by direct injection. The ex-vivo method is described in the second half of the review. Attempts have been made to use this therapy for bone, cartilage, wound, urothelial, nerve tissue regeneration and for treating diabetes using viral or non-viral vectors. Again porous polymers can be used as scaffolds for cell transplantation. There are as yet few comparisons between these many different variables to show which is the best for any particular application. With few exceptions, all of the results were positive in showing some gene expression and some consequent effect on tissue growth and remodelling. Some of the principal advantages and disadvantages of various methods are discussed.

Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Delta cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

Recent advances in ocular gene therapy

Ocular gene therapy has made significant advances due to improvements in viral vector delivery methods. Recent clinical trials for the treatment of Leber's congenital amaurosis have provided evidence for successful in-vivo gene therapy in humans. Gene therapy for ocular disease has been under investigation just over 15 years. Recently, the first human gene therapy trials for retinal degeneration were undertaken with encouraging preliminary safety and efficacy findings. Building on previous success in reversing blindness in animal models of Leber's congenital amaurosis, several groups proceeded with adeno-associated virus-mediated gene replacement. Many of the humans demonstrated increases in light sensitivity and in visual acuity. Subjective improvements in vision were corroborated in some cases by objective tests such as pupillary light response and nystagmography. Although much of the work in ocular gene therapy has involved retinal applications, significant progress has been seen in other aspects of ophthalmology. Ongoing human clinical trials support the safety and efficacy of adeno-associated virus-mediated gene therapy for retinal disease. These and other studies will establish the foundation for methodology to treat additional ocular diseases using gene therapy strategies.

In Vivo Functional and Anatomic Imaging for Assessment of in Vivo Gene Transfer

To assess whether a combination of in vivo anatomic and functional imaging can help quantify expression of somatostatin receptor type 2 (SSTR2)-based reporters after in vivo gene transfer. All animal experiments were approved by an institutional animal care and use committee. Six nude mice bearing two subcutaneous L3.6pl (human pancreatic cancer) tumors were injected intratumorally with an adenovirus containing a human somatostatin receptor type 2 gene chimera (Ad-HA-SSTR2) or a control adenovirus containing green fluorescent protein (Ad-GFP). Two days later, magnetic resonance (MR) imaging was performed to derive tumor weight and analyze morphology. Intravenous injection of Food and Drug Administration-approved indium 111 octreotide was followed by gamma camera imaging (planar imaging and single photon emission computed tomography [SPECT]) the next day. Region-of-interest analysis followed. The procedure was also performed in six nude mice with slow-growing MDA-MB-435 (human breast carcinoma) tumors, which allowed serial imaging 3 days and 2 weeks after adenovirus injection. After imaging, excised tumor weight and biodistribution were assessed. Statistical analyses included a Student t test and linear regression. With both tumor types, ex vivo and image-based in vivo biodistribution demonstrated greater uptake (percentage of injected dose per gram) in tumors infected with Ad-HA-SSTR2 than in those infected with Ad-GFP (P < .05). Furthermore, in vivo and ex vivo biodistribution analysis correlated (ex vivo vs planar and MR imaging: r = 0.87, P < .05, n = 24; ex vivo vs SPECT and MR imaging: r = 0.84, P < .05, n = 24). Moreover, in vivo biodistribution distinguished greater expression at the earlier time point in MDA-MB-435 tumors infected with Ad-HA-SSTR2 from waning expression at the later time point (P < .05). A combination of in vivo functional and anatomic imaging methods can help quantify gene expression after in vivo gene transfer.

Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line.

BackgroundThe Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases.ResultsThe tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency.ConclusionThe tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal maculopathies.

S1752 The Interleukin (IL)-10 Gene Is Associated with Crohn's Disease in Canadian Children. Findings Based On a Gene-Wide Case-Control Study

Background and Aim: RNAi is a powerful biological tool whereby any specific genes of interest can be specifically targeted. However, application of RNAi for silencing genes of the GI tract has proven difficult. We recently described a bacteria-based RNAi technology (transkingdom RNAi, tkRNAi) to simultaneously produce and deliver RNAi to the GI tract (Xiang, Nat Biotech 2006, 697). The aim of this study was to examine whether tkRNAi can be used to silence the expression of IL-10, an anti-inflammatory cytokine, and induce a pro-inflammatory state In Vivo. Methods: After selection of themost powerful siRNA sequence, tkRNAi bacteria were created producing shRNA against mouse IL-10 (TRIP-sh7) and control (TRIP-scrambled). J774.A1 cells were incubated with TRIP-bearing bacteria for 2h at various MOIs, and then stimulated with LPS for 24h. IL-10 gene silencing was analyzed by quantitative RT-PCR and ELISA. To assess In Vivo gene silencing, 12 Balb/c mice were randomized into 3 groups: untreated (n=4); TRIP-sh7 (n=4); TRIP-scrambled (n=4). Following bacteria treatment for 2 weeks, colons were harvested and analyzed by qRT-PCR and IHC staining for IL-10 and γ-IFN. Results: treatment of J774.A1 cells with TRIP-sh7 reduced IL-10 mRNA and protein levels by >80% (p 70% (p < 0.05) compared to animals treated with TRIPscrambled control bacteria. IHC staining of colon tissue demonstrated a marked reduction of IL-10 protein expression in TRIP-sh7 treated mice compared to untreated or mice treated with control bacteria. Moreover, treatment with TRIP-sh7 was associated with increased colonic production of the TH1 cytokine γ-IFN. Treatment with TRIP-sh7 for two weeks was also associated with a significant loss of body weight compared to untreated or mice treated with control bacteria (p < 0.05 for both). Summary and Conclusion: bacteria expressing shRNA against IL-10 can mediate potent gene silencing in LPS-stimulated J774.A1 cells, and in colonic epithelial cells In Vivo. Furthermore colonic levels of γ-IFN were markedly increased following bacterial RNAi against IL-10. tkRNAi approach may be useful for In Vivo functional genomics studies in the GI tract, and for developing RNAi therapies for GI diseases. Since tkRNAi can be adapted to induce silencing in multiple animal species, this approach may be suited for development of animal models in rodents or non-rodent, for diseases such as IBD.

523. Non-Viral Phi C31 Integrase Mediated In Vivo Gene Delivery to Adult Murine Retinal Pigment Epithelial Cells

523. Non-Viral Phi C31 Integrase Mediated In Vivo Gene Delivery to Adult Murine Retinal Pigment Epithelial Cells

163. Non-Viral In Vivo Gene Transfer Confers Long Term Transgene Expression

163. Non-Viral In Vivo Gene Transfer Confers Long Term Transgene Expression

S1751 Copy Number Variant and Extended SNP Genome Wide Association Study in Celiac Disease

Background and Aim: RNAi is a powerful biological tool whereby any specific genes of interest can be specifically targeted. However, application of RNAi for silencing genes of the GI tract has proven difficult. We recently described a bacteria-based RNAi technology (transkingdom RNAi, tkRNAi) to simultaneously produce and deliver RNAi to the GI tract (Xiang, Nat Biotech 2006, 697). The aim of this study was to examine whether tkRNAi can be used to silence the expression of IL-10, an anti-inflammatory cytokine, and induce a pro-inflammatory state In Vivo. Methods: After selection of themost powerful siRNA sequence, tkRNAi bacteria were created producing shRNA against mouse IL-10 (TRIP-sh7) and control (TRIP-scrambled). J774.A1 cells were incubated with TRIP-bearing bacteria for 2h at various MOIs, and then stimulated with LPS for 24h. IL-10 gene silencing was analyzed by quantitative RT-PCR and ELISA. To assess In Vivo gene silencing, 12 Balb/c mice were randomized into 3 groups: untreated (n=4); TRIP-sh7 (n=4); TRIP-scrambled (n=4). Following bacteria treatment for 2 weeks, colons were harvested and analyzed by qRT-PCR and IHC staining for IL-10 and γ-IFN. Results: treatment of J774.A1 cells with TRIP-sh7 reduced IL-10 mRNA and protein levels by >80% (p 70% (p < 0.05) compared to animals treated with TRIPscrambled control bacteria. IHC staining of colon tissue demonstrated a marked reduction of IL-10 protein expression in TRIP-sh7 treated mice compared to untreated or mice treated with control bacteria. Moreover, treatment with TRIP-sh7 was associated with increased colonic production of the TH1 cytokine γ-IFN. Treatment with TRIP-sh7 for two weeks was also associated with a significant loss of body weight compared to untreated or mice treated with control bacteria (p < 0.05 for both). Summary and Conclusion: bacteria expressing shRNA against IL-10 can mediate potent gene silencing in LPS-stimulated J774.A1 cells, and in colonic epithelial cells In Vivo. Furthermore colonic levels of γ-IFN were markedly increased following bacterial RNAi against IL-10. tkRNAi approach may be useful for In Vivo functional genomics studies in the GI tract, and for developing RNAi therapies for GI diseases. Since tkRNAi can be adapted to induce silencing in multiple animal species, this approach may be suited for development of animal models in rodents or non-rodent, for diseases such as IBD.

S1750a Silencing of Interleukin-10 in the Gastrointestinal Tract Using Transkingdom RNA Interference In Vivo

Background and Aim: RNAi is a powerful biological tool whereby any specific genes of interest can be specifically targeted. However, application of RNAi for silencing genes of the GI tract has proven difficult. We recently described a bacteria-based RNAi technology (transkingdom RNAi, tkRNAi) to simultaneously produce and deliver RNAi to the GI tract (Xiang, Nat Biotech 2006, 697). The aim of this study was to examine whether tkRNAi can be used to silence the expression of IL-10, an anti-inflammatory cytokine, and induce a pro-inflammatory state In Vivo. Methods: After selection of themost powerful siRNA sequence, tkRNAi bacteria were created producing shRNA against mouse IL-10 (TRIP-sh7) and control (TRIP-scrambled). J774.A1 cells were incubated with TRIP-bearing bacteria for 2h at various MOIs, and then stimulated with LPS for 24h. IL-10 gene silencing was analyzed by quantitative RT-PCR and ELISA. To assess In Vivo gene silencing, 12 Balb/c mice were randomized into 3 groups: untreated (n=4); TRIP-sh7 (n=4); TRIP-scrambled (n=4). Following bacteria treatment for 2 weeks, colons were harvested and analyzed by qRT-PCR and IHC staining for IL-10 and γ-IFN. Results: treatment of J774.A1 cells with TRIP-sh7 reduced IL-10 mRNA and protein levels by >80% (p 70% (p < 0.05) compared to animals treated with TRIPscrambled control bacteria. IHC staining of colon tissue demonstrated a marked reduction of IL-10 protein expression in TRIP-sh7 treated mice compared to untreated or mice treated with control bacteria. Moreover, treatment with TRIP-sh7 was associated with increased colonic production of the TH1 cytokine γ-IFN. Treatment with TRIP-sh7 for two weeks was also associated with a significant loss of body weight compared to untreated or mice treated with control bacteria (p < 0.05 for both). Summary and Conclusion: bacteria expressing shRNA against IL-10 can mediate potent gene silencing in LPS-stimulated J774.A1 cells, and in colonic epithelial cells In Vivo. Furthermore colonic levels of γ-IFN were markedly increased following bacterial RNAi against IL-10. tkRNAi approach may be useful for In Vivo functional genomics studies in the GI tract, and for developing RNAi therapies for GI diseases. Since tkRNAi can be adapted to induce silencing in multiple animal species, this approach may be suited for development of animal models in rodents or non-rodent, for diseases such as IBD.

S1754 Interactions Between PTPN2, CARD15 and ATG16L1 Genes Are Associated with Pediatric-Onset Crohn's Disease. Findings Based On a Gene-Wide Case-Control Study in Canadian Children

Background and Aim: RNAi is a powerful biological tool whereby any specific genes of interest can be specifically targeted. However, application of RNAi for silencing genes of the GI tract has proven difficult. We recently described a bacteria-based RNAi technology (transkingdom RNAi, tkRNAi) to simultaneously produce and deliver RNAi to the GI tract (Xiang, Nat Biotech 2006, 697). The aim of this study was to examine whether tkRNAi can be used to silence the expression of IL-10, an anti-inflammatory cytokine, and induce a pro-inflammatory state In Vivo. Methods: After selection of themost powerful siRNA sequence, tkRNAi bacteria were created producing shRNA against mouse IL-10 (TRIP-sh7) and control (TRIP-scrambled). J774.A1 cells were incubated with TRIP-bearing bacteria for 2h at various MOIs, and then stimulated with LPS for 24h. IL-10 gene silencing was analyzed by quantitative RT-PCR and ELISA. To assess In Vivo gene silencing, 12 Balb/c mice were randomized into 3 groups: untreated (n=4); TRIP-sh7 (n=4); TRIP-scrambled (n=4). Following bacteria treatment for 2 weeks, colons were harvested and analyzed by qRT-PCR and IHC staining for IL-10 and γ-IFN. Results: treatment of J774.A1 cells with TRIP-sh7 reduced IL-10 mRNA and protein levels by >80% (p 70% (p < 0.05) compared to animals treated with TRIPscrambled control bacteria. IHC staining of colon tissue demonstrated a marked reduction of IL-10 protein expression in TRIP-sh7 treated mice compared to untreated or mice treated with control bacteria. Moreover, treatment with TRIP-sh7 was associated with increased colonic production of the TH1 cytokine γ-IFN. Treatment with TRIP-sh7 for two weeks was also associated with a significant loss of body weight compared to untreated or mice treated with control bacteria (p < 0.05 for both). Summary and Conclusion: bacteria expressing shRNA against IL-10 can mediate potent gene silencing in LPS-stimulated J774.A1 cells, and in colonic epithelial cells In Vivo. Furthermore colonic levels of γ-IFN were markedly increased following bacterial RNAi against IL-10. tkRNAi approach may be useful for In Vivo functional genomics studies in the GI tract, and for developing RNAi therapies for GI diseases. Since tkRNAi can be adapted to induce silencing in multiple animal species, this approach may be suited for development of animal models in rodents or non-rodent, for diseases such as IBD.

143. Non-Viral Phi C31 Integrase Mediated In Vivo Gene Delivery to the Adult Murine Kidney

143. Non-Viral Phi C31 Integrase Mediated In Vivo Gene Delivery to the Adult Murine Kidney

In-vivo gene transfer induces transgene expression in cells and secretions of the mouse cauda epididymis

Mouse cauda epididymis were in-vivo transfected using the lipid FuGENE 6 as gene vector. Two gene constructions were employed: the p-GeneGRIP which codifies for the Green Fluorescent Protein (GFP) and the pSEAP-control that expresses an alkaline phosphatase as a secretion. Transfection was detected by fluorescence and appeared in the nucleus and cytoplasm of epithelial cells. Transfection was observed in 39.70% of cells after 2 days and in 31.77% after 7 days, and then diminished progressively. Moreover, the presence of the transgene in the DNA isolated from treated epididymides was observed by polymerase chain reaction. GFP gene expression appeared in large areas of the cauda epididymis and it was observed exclusively in the cytoplasm of epithelial cells. GFP gene expression occurred during 2 weeks after gene injection and occupied 32.24, 29.98 and 22.37% of the area of the tubules when analyzed 2, 7 and 15 days after gene injection. The cauda was also analyzed in toto and showed similar results. The use of the pSEAP-control gene showed that cauda epididymis secretions can also be modified by the transfection procedure. A significant increase of alkaline phosphatase activity appeared in the epididymal fluids 7 days after gene injection. These results indicate that transfection procedures could be an important tool in the future to study epididymal physiology or to change the fertilizing ability of spermatozoa.