scholarly journals Hypoxia-inducible expression of BAX: application in tumor-targeted gene therapy

2000 ◽  
Vol 8 (4) ◽  
pp. 1-7 ◽  
Author(s):  
Hangjun Ruan ◽  
Lily Hu ◽  
Jingli Wang ◽  
Tomoko Ozawa ◽  
Nader Sanai ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Expression System ◽  
Fluorescent Microscopy ◽  
Hypoxia Inducible Factor ◽  
Specific Gene ◽  
Gfp Expression ◽  
Transcriptional Complex

Object The presence of hypoxic cells in human brain tumors contributes to the resistance of these tumors to radiation therapy. However, because normal tissues are not hypoxic, the presence of hypoxic cells also provides the potential for designing cancer-specific gene therapy. Suicide genes can be expressed specifically in hypoxic conditions by hypoxia-responsive elements (HREs), which are activated through the transcriptional complex hypoxia-inducible factor–1 (HIF-1). Methods The authors have transfected the murine BAX–green fluorescent protein (GFP) fusion gene under the regulation of three copies of HRE into U-87 MG and U-251 MG cells and selected stably transfected clones. Even though BAX was expressed under both oxic and anoxic conditions in these clones, cell survival assays demonstrated increased cell killing under anoxic as compared with oxic conditions. Cells obtained from most of these clones did not grow in vivo, or the tumors exhibited highly variable growth rates. However, cells obtained from the U-251 MG clone A produced tumors that grew as well as tumors derived from parental cells, and examination of the tumor sections under fluorescent microscopy revealed GFP expression in localized regions. Western blot analyses confirmed an increased BAX expression in these tumors. Analysis of the results suggests that HRE-regulated BAX can be a promising tool to target hypoxic brain tumor cells. However, there are measurable levels of BAX-GFP expression in this three-copy HRE–mediated expression system under oxia, suggesting promoter leakage. In addition, most clones did not show significant induction of BAX-GFP under anoxia. Therefore, the parameters of this HRE-mediated expression system, including HRE copy number and the basal promoter, need to be optimized to produce preferential and predictable gene expression in hypoxic cells.

scholarly journals Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christina L. Parker ◽  
Timothy M. Jacobs ◽  
Justin T. Huckaby ◽  
Dimple Harit ◽  
Samuel K. Lai
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Endosomal Escape ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Specific Gene ◽  
Targeted Gene Delivery

ABSTRACT Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo. We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2−) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy. IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

Abstract 38: Rbp-j Regulates the Genetic Program of the Myo-epithelioid JG Cell

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Ruth M Castellanos Rivera ◽  
Ellen S. Pentz ◽  
Kenneth W. Gross ◽  
Silvia Medrano ◽  
Jing Yu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Fluorescent Protein ◽  
Smooth Muscle Actin ◽  
Artificial Chromosome ◽  
Specific Protein ◽  
Genetic Program ◽  
Cell Phenotype ◽  
Gfp Expression ◽  
Renin Gene

RBP-J , the major downstream effector of Notch signaling, is necessary to maintain the number of juxtaglomerular (JG) cells. In addition, RBP-J regulates the plasticity of arteriolar smooth muscle cells to adopt the renin cell phenotype when homeostasis is threatened. We hypothesized that RBP-J acts as an on/off switch controlling the expression of genes that determine the renin phenotype. To determine whether RBP-J directly affects renin gene expression, we generated mice harboring a bacterial artificial chromosome (BAC) transgene with green fluorescent protein (GFP) under the control of the renin gene carrying a mutation in its RBP-J- binding site (Mut-BAC). Mut-BAC mice had markedly reduced GFP expression to 12.9 % ±0.01 (n=3) of the control (Wt-BAC) and a diminished response to homeostatic challenges: mut-BAC mice had a reduced number of GFP positive JG areas per total number of glomeruli (Wt-BAC: 25.1 % ±3.0, n=3; Mut-BAC: 9.3 % ±1.4, n=2, p<0.02) and no GFP expression along the arterioles. To determine whether the decrease in the number of JG cells in mice lacking RBP-J (cKO) was due to a diminished endowment of renin progenitor cells, we traced the fate of cells derived from the renin lineage by generating mice ( RBP-J fl/fl ; Ren1d +/cre ; R26R +/- ) in which cells lacking RBP-J simultaneously expressed β-galactosidase (β-gal). The pattern of β-gal in cKO and control kidneys was identical, indicating that cells derived from the renin lineage did not die but instead changed their phenotype. Next we investigated the phenotype adopted by the cells derived from the renin lineage. Expression of α-smooth muscle actin and smoothelin (a marker of mature smooth muscle) was significantly decreased to 41 % ±7.0 (n=2) and 44 % ±8.8 (n=2) respectively with respect to controls (p<0.01). In addition, mutant JG cells in vivo did not express genes characteristic of the renin phenotype such as renin, calponin1, Nfat and Akr1b7 expressing instead fibroblast-specific protein 1 indicating the adoption of a fibroblast-like phenotype. Results indicate that RBP-J directly governs a genetic program that controls the dual endocrine-contractile phenotype of the JG cell, which is crucial to maintain blood pressure and fluid-electrolyte homeostasis.

scholarly journals Intra-testicular injection of adenoviral constructs results in Sertoli cell-specific gene expression and disruption of the seminiferous epithelium

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 361-370 ◽  
Author(s):  
R P Hooley ◽  
M Paterson ◽  
P Brown ◽  
K Kerr ◽  
P T K Saunders
Keyword(s):  
Seminiferous Tubule ◽  
Fluorescent Protein ◽  
Immune Cell ◽  
Adenoviral Vectors ◽  
Seminiferous Tubules ◽  
Specific Gene ◽  
Viral Particles ◽  
Junctional Complexes ◽  
Testicular Injection

Spermatogenesis is a complex process that cannot be modelledin vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-downin vivo. We describe here a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC functionin vivoand future work will therefore focus on the use of lentiviral delivery systems.

6 Invitro validation of gene edited phenotypes using CRISPR-dCas9 transcriptional activators

2020 ◽  
Vol 32 (2) ◽  
pp. 127
Author(s):  
K. M. Polkoff ◽  
N. K. Gupta ◽  
J. A. Piedrahita
Keyword(s):  
Dna Sequences ◽  
Fluorescent Protein ◽  
Transcriptional Start Site ◽  
Fluorescent Microscopy ◽  
Large Animal ◽  
Transcriptional Activators ◽  
Gfp Expression ◽  
Time Investment ◽  
Donor Cells ◽  
Fetal Fibroblasts

Despite the extensive advantages of gene-edited large animals for agriculture and biomedical purposes, they represent a large monetary and time investment due to high husbandry costs, long gestation lengths, and few offspring; that is, 9 months for one calf and almost 4 months for pigs. Even with known DNA sequences before somatic cell nuclear transfer (SCNT), inserted transgenes are often not expressed as expected. Therefore, there is a need to phenotypically validate the gene modifications invitro before investing time and resources in the creation of a gene-edited large animal; however, many gene targets are tissue specific and not expressed in SCNT donor cells. In this work, we show that CRISPR-dCas9 transcriptional activators (TAs) can be used to validate functional transgene insertion in nonexpressing SCNT donor cells, in our case fetal fibroblasts. To demonstrate this concept, we first generated a DNA knockin of the H2B-GFP sequence into the porcine LGR5 locus. CRISPR/Cas9 nuclease was used to create a double-stranded break in the genomic DNA downstream of the LGR5 promoter. A homology-directed repair template plasmid containing H2B-GFP flanked by 1000bp homology arms flanking the cut site was co-transfected with the Cas9 and gRNA, and cells were seeded at low density for colony outgrowth. Colonies were genotyped by PCR and sequencing to verify successful targeted transgene integration. To test whether TAs allow for invitro validation of transgene expression, 5×105 wildtype or gene-edited fibroblasts were nucleofected (Lonza) with 500ng total of four gRNA plasmids (Addgene #43860) designed to target the 1-kb region upstream of the LGR5 transcriptional start site in combination with 500ng VP64-dCas9 (Addgene #47107). Detection of green fluorescent protein (GFP) was analysed by fluorescent microscopy followed by flow cytometry; at least 30 000 events were recorded for each treatment (Cytoflex). Our results show that GFP was detected in on average 28.6% of the gene-edited cells transfected with LGR5 TAs but not detected in gene-edited cells that were not transfected with LGR5 TAs (0%) or in wild-type cells transfected with the LGR5 TAs (0%). The experiment was repeated three times. Next, to prove that our invitro validation replicates the invivo phenotype, the gene-edited colonies heterozygous for the insertion were used for SCNT to generate piglets. Epidermal cells, which contain a population of LGR5+ stem cells, were isolated from the skin and sorted for GFP expression. The RT-qPCR results from GFP+ or GFP− cells showed the presence of LGR5 transcripts in the GFP+ cells but not GFP− cells. In conclusion, TAs were necessary and sufficient to detect LGR5-promoter driven H2B-GFP expression in gene-edited fibroblasts invitro, which faithfully recapitulates the invivo phenotype of the gene-edited animal. Further preliminary data from our laboratory suggest that our novel method can be used to detect successful gene knockouts in addition to transgene knockins and can be used to validate phenotypic outcomes of DNA modifications before the generation of gene-edited animals.

scholarly journals Effect of mechanical stretch on HIF-1α and MMP-2 expression in capillaries isolated from overloaded skeletal muscles: laser capture microdissection study

2005 ◽  
Vol 289 (3) ◽  
pp. H1315-H1320 ◽  
Author(s):  
Malgorzata Milkiewicz ◽  
Tara L. Haas
Keyword(s):  
Endothelial Cells ◽  
Laser Capture Microdissection ◽  
Skeletal Muscles ◽  
Hypoxia Inducible Factor ◽  
Fold Increase ◽  
Mechanical Stretch ◽  
Specific Gene ◽  
Capillary Endothelial Cells ◽  
Laser Capture

Under physiological nonhypoxic conditions, angiogenesis can be driven by mechanical forces. However, because of the limitations of the specific gene expression analysis of microvessels from in vivo experiments, the mechanisms regulating the coordinated expression of angiogenic factors implicated in the process remain intangible. In this study, the technique of laser capture microdissection (LCM) was adapted for the study of angiogenesis in skeletal muscles. With a combination of LCM and real-time quantitative PCR it was demonstrated that capillary endothelial cells produce matrix metalloproteinase (MMP)-2 and that mechanical stretch of capillaries within muscle tissue markedly increases MMP-2 mRNA (2.5-fold increase vs. control; P < 0.05). In addition, we showed that transcription factor hypoxia-inducible factor (HIF)-1α expression was 13.5-fold higher in capillaries subjected to stretch compared with controls ( P < 0.05). These findings demonstrate the feasibility of this approach to study angiogenic gene regulation and provide novel evidence of HIF-1α induction in stretched capillary endothelial cells.

scholarly journals Imaging Expression of Cytosine Deaminase-Herpes Virus Thymidine Kinase Fusion Gene (CD/TK) Expression with [124I]FIAU and PET

2002 ◽  
Vol 1 (1) ◽  
pp. 153535002002000 ◽  
Author(s):  
Trevor Hackman ◽  
Michail Doubrovin ◽  
Julius Balatoni ◽  
Tatiana Beresten ◽  
Vladimir Ponomarev ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Thymidine Kinase ◽  
Enzyme Assay ◽  
Fusion Gene ◽  
Cytosine Deaminase ◽  
Prodrug Activation ◽  
Wide Range ◽  
Different Levels

Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)herpes simplex virus type 1 thymidine kinase ( HSV1-tk) fusion gene ( CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.

scholarly journals In vivo analysis of key elements within the renin regulatory region

2008 ◽  
Vol 35 (3) ◽  
pp. 243-253 ◽  
Author(s):  
Sean T. Glenn ◽  
Craig A. Jones ◽  
Li Pan ◽  
Kenneth W. Gross
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Region ◽  
Renin Angiotensin System ◽  
Artificial Chromosome ◽  
Proximal Promoter ◽  
Gfp Expression ◽  
Renin Gene ◽  
Green Fluorescent

Renin is responsible for initiating the enzymatic cascade that results in the production of angiotensin II, the major effector molecule of the renin-angiotensin system (RAS). Extensive information on the regulatory region of the renin gene has been derived by transient transfection studies in vitro, particularly using the As4.1 cell line. To verify key factors within the regulatory region of renin in vivo, homologous recombination was used to introduce a green fluorescent protein (GFP) cassette into exon one of the renin gene contained within a 240 kb bacterial artificial chromosome (BAC) to create a construct that has GFP expression controlled by the renin regulatory region (RenGFP BAC). Within the regulatory region of the RenGFP BAC construct we independently deleted the enhancer, as well as mutated the HOX-PBX site within the proximal promoter element. Transgenic lines were generated for each of these BAC constructs and GFP expression was analyzed throughout a spectrum of tissues positive for renin expression including the kidney, adrenal gland, gonadal artery, and submandibular gland. The results described within this manuscript support the interpretation that the renin enhancer is critical for regulating baseline expression where as the Hox/Pbx site is important for the tissue specificity of renin expression.

scholarly journals Development of a Tetracycline-Inducible Gene Expression System for the Study of Helicobacter pylori Pathogenesis

2013 ◽  
Vol 79 (23) ◽  
pp. 7351-7359 ◽  
Author(s):  
Aleksandra W. Debowski ◽  
Phebe Verbrugghe ◽  
Miriam Sehnal ◽  
Barry James Marshall ◽  
Mohammed Benghezal
Keyword(s):  
Helicobacter Pylori ◽  
Animal Models ◽  
Chronic Infection ◽  
Fluorescent Protein ◽  
Expression System ◽  
Content Type ◽  
Conditional Expression ◽  
H Pylori ◽  
Green Fluorescent

ABSTRACTDeletion mutants and animal models have been instrumental in the study ofHelicobacter pyloripathogenesis. Conditional mutants, however, would enable the study of the temporal gene requirement duringH. pyloricolonization and chronic infection. To achieve this goal, we adapted theEscherichia coliTn10-derived tetracycline-inducible expression system for use inH. pylori. TheureApromoter was modified by inserting one or twotetoperators to generate tetracycline-responsive promoters, nameduPtetO, and these promoters were then fused to the reportergfpmut2 and inserted into different loci. The expression of the tetracycline repressor (tetR) was placed under the control of one of three promoters and inserted into the chromosome. Conditional expression of green fluorescent protein (GFP) in strains harboringtetRanduPtetO-GFPwas characterized by measuring GFP activity and by immunoblotting. The twotet-responsiveuPtetOpromoters differ in strength, and induction of these promoters was inducer concentration and time dependent, with maximum expression achieved after induction for 8 to 16 h. Furthermore, the chromosomal location of theuPtetO-GFPconstruct and the nature of the promoter driving expression oftetRinfluenced the strength of theuPtetOpromoters upon induction. Integration ofuPtetO-GFPandtetRconstructs at different genomic loci was stablein vivoand did not affect colonization. Finally, we demonstrate tetracycline-dependent induction of GFP expressionin vivoduring chronic infection. These results open new experimental avenues for dissectingH. pyloripathogenesis using animal models and for testing the roles of specific genes in colonization of, adaptation to, and persistence in the host.

scholarly journals OTX5 Regulates Pineal Expression of the Zebrafish REV-ERBα through a New DNA Binding Site

2008 ◽  
Vol 22 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Shin-ichi Nishio ◽  
Tomoko Kakizawa ◽  
Gilles Chatelain ◽  
Gérard Triqueneaux ◽  
Frédéric Brunet ◽  
...  
Keyword(s):  
Pineal Gland ◽  
Binding Site ◽  
Fluorescent Protein ◽  
Specific Gene ◽  
Orphan Nuclear Receptor ◽  
Cis Acting ◽  
Lower Vertebrates ◽  
Green Fluorescent ◽  
Zebrafish Embryogenesis

Abstract The pineal gland plays a central role in the photoneuroendocrine system and acts as a photosensory organ in lower vertebrates. The orphan nuclear receptor Rev-erbα (NR1D1) has previously been shown to be expressed in the pineal and to be regulated with a robust circadian rhythm during zebrafish embryogenesis. This early pineal expression is under the control of the transcription factor Orthodenticle homeobox 5 (Otx5). In this paper, we show that Otx5 regulates the second zfRev-erbα promoter, ZfP2. Despite the absence of a classical Otx-binding site within ZfP2, this regulation depends on the integrity of the Otx5 homeodomain. Mapping experiments as well as EMSAs show that this interaction between Otx5 and ZfP2 depends on a noncanonical bipartite Otx-binding site (GANNCTTA and TAAA) that we called pineal expression related element (PERE). We showed that PERE is necessary for pineal expression in vivo by injecting zebrafish embryos with wild type and mutated versions of zfRev-erbα promoter fused to green fluorescent protein. Interestingly, PERE is found upstream of other genes expressed in the pineal gland, suggesting that it may play an important role in governing pineal expression. Our data establish that PERE is a novel cis-acting element contributing to pineal-specific gene expression and to Otx target gene regulation.

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