Antisense modulation of the coding or regulatory sequence of the folate receptor (folate binding protein-1) in mouse embryos leads to neural tube defects

BACKGROUND

Although folic acid decreases the incidence of neural tube defects (NTDs) in humans, the mechanism for this protection is unknown. We have employed antisense technology to alter expression of the gene for the folate receptor (folate binding protein‐1 [Folbp1]) in mouse embryos cultured in vitro.

METHODS

Embryos were explanted on day 8 of gestation and cultured for 44 hr. Several oligodeoxyribonucleotides designed to modulate the coding region or a regulatory sequence in the 5′‐untranslated region of Folbp1 were microinjected into the amniotic sac of embryos at the beginning of the culture period.

RESULTS

Two different antisense sequences to the 5′ and 3′ coding region in Folbp1 produced concentration‐dependent increases in the number of embryos with NTDs. Coinjection of 5‐methyltetrahydrofolate with these sequences decreased the frequency of abnormal embryos. A semi‐quantitative RT‐PCR technique used to measure the amount of Folbp1 mRNA in treated and control embryos confirmed that the mRNA level was decreased by treatment with the antisense sequences. An antisense oligodeoxyribonucleotide to a 17 base cis regulatory element also generated a concentration‐dependent increase in the frequency of embryos with NTDs, and a decrease in the level of Folbp1 mRNA.

CONCLUSIONS

These results demonstrate that alterations in expression of Folbp1 by perturbing either the coding sequence or a critical regulatory cis‐element can play a role in NTDs. Birth Defects Research (Part A) 67475–487, 2003. © 2003 Wiley‐Liss, Inc.

     

Somatic gene transfer into the lactating ovine mammary gland

Background

Somatic gene therapy requires safe and efficient techniques for the gene transfer procedure. The ovine mammary gland is described as a model system for the evaluation of somatic gene transfer methods.

Methods

Different gene delivery formulations were retrogradely injected into the mammary gland of lactating sheep. The efficiency of the gene transfer was subsequently measured by the detection of the secreted transgene products in the milk. To counteract the milk flow in the lactating gland caused by the permanent milk production, a newly developed pretreatment of the mammary gland with hyperosmotic solutions was applied. In addition, in vivo electroporation of DNA into the mammary gland is described.

Results

Gene transfer using naked DNA or simple complexes of DNA with polycations did not result in traceable amounts of reporter gene products. However, utilizing the complex cationic lipid DOSPER, a peak expression of about 400 ng/ml was observed 6 days after transfection. Maximum expression rates of more than 1 µg/ml were obtained by combining hyperosmotic pretreatment and receptor‐mediated gene transfer. For the in vivo electroporation, the proof of principle for this technique in the mammary gland is reported.

Conclusions

The ovine mammary gland turned out to be a very well suited as a model system for evaluation and optimization of various gene transfer protocols. Copyright © 2002 John Wiley & Sons, Ltd.

     

The use of low-molecular-weight PEIs as gene carriers in the monkey fibroblastoma and rabbit smooth muscle cell cultures

Background

Polyethylenimines (PEIs) and cationic polymers have been used successfully in gene delivery. In earlier reports, only large PEIs (MW>10 000) have shown significant transfection efficiency. In the present study, the roles of small PEIs (MW 700 and 2000) were studied as additional compounds to see if they can improve gene delivery with cationic liposomes.

Methods

The TKBPVlacZ expression plasmid was transfected in the CV1‐P (monkey fibroblastoma) and SMC (rabbit smooth muscle) cell lines using various combinations of PEIs (MW 700, 2000, and 25 000) and Dosper liposomes. The transfection efficiency was determined with the fluorometric ONPG (o‐nitrophenol‐β‐D ‐galactopyranoside) assay and histochemical X‐gal staining. The toxicity of the transfection reagents was estimated by the MTT [3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyl tetrazolium bromide] assay.

Results

Transfection of TKBPVlacZ plasmid by the small PEIs (MW 700 and 2000) combined with Dosper liposomes was associated with high expression of the lacZ reporter gene in the CV1‐P and SMC cell lines. The transfection efficiencies of the low‐molecular‐weight PEI/liposome combinations were several fold higher than those of PEIs or liposomes alone. PEI/liposome combinations had no toxicity on the cell lines tested.

Conclusions

The low‐molecular‐weight PEIs could be used successfully for gene delivery when combined with the cationic liposomes, resulting in a synergistic increase of the transfection efficiency in both cell lines studied. Copyright © 2002 John Wiley & Sons, Ltd.

     

Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells

Background

Several approaches for gene therapy of cystic fibrosis using viral and non‐viral vectors are currently being undertaken. Nevertheless, the present data suggest that vectors currently being used will either have to be further modified or, alternatively, novel vector systems need to be developed. Recently, bacteria have been proven as suitable vehicles for DNA transfer to a wide variety of eukaryotic cells. In this study, we assessed the ability of the facultative intracellular pathogen Listeria monocytogenes to deliver a cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) to CHO‐K1 cells, since these cells have been extensively used for heterologous CFTR expression.

Methods

An established in vitro gene transfer system based on antibiotic‐mediated lysis of intracellular L. monocytogenes was exploited to transfer eukaryotic expression plasmids. Transient as well as stable CFTR transgene expression was analyzed by microscopical and biochemical methods; functionality was tested by whole‐cell patch‐clamp recordings.

Results

L. monocytogenes mediated gene transfer to CHO‐K1 cells was facilitated by an improved transfection protocol. In addition, the use of the isogenic mutant L. monocytogenes hlyW491A, engineered to produce a hemolysin variant with low toxigenic activity, greatly enhanced the efficiency of gene transfer. This strain allowed the transfer of functional CFTR to CHO‐K1 cells.

Conclusions

This is the first demonstration of L. monoyctogenes mediated CFTR transgene transfer. The successful in vitro transfer suggests that L. monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system

Background

Gene therapy strategies for the treatment of vascular disease such as the prevention of post‐angioplasty restenosis require efficient, non‐toxic transfection of vascular cells. In vitro studies in these cells contribute to vector development for in vivo use and for the evaluation of genes with therapeutic potential. The aim of this project was to evaluate a novel synthetic vector consisting of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), which combine to form the LID vector complex.

Methods

Cultures of porcine smooth muscle cells and endothelial cells were established and then transfected with the LID vector, using the reporter genes luciferase and green fluorescent protein and the metalloprotease inhibitor TIMP‐1.

Results

The LID vector system transfected primary porcine vascular smooth muscle cells and porcine aortic endothelial cells with efficiency levels of 40% and 35%, respectively. By increasing the relative DNA concentration four‐fold, incubation periods as short as 30 min achieved the same levels of luciferase transgene expression as 4 h incubations at lower DNA concentrations. The transfection did not affect cell viability as measured by their proliferative potential. Serum levels of up to 20% in the transfection medium had no adverse affect on the efficiency of transfer and gene expression in either cell type. Transfections with the cDNA for TIMP‐1 produced protein levels that peaked at 130 ng/ml per 24 h and persisted for 14 days at 10 ng/ml per 24 h.

Conclusion

This novel vector system has potential for studies involving gene transfer to cardiovascular cells in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectors

Background

Lentiviral vectors allow gene transfer into non‐dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing.

Methods

To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon‐optimized gag‐pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV‐G) under the control of an ponasterone‐inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized.

Results

The RT activity and vector titers of cell clones stably transfected with the inducible gag‐pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone‐inducible VSV‐G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone‐induced producer clones vector titers of more than 1×10⁵ transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production.

Conclusions

The packaging cells described should be suitable for most preclinical applications of SIV‐based vectors. By avoiding regions of high homology between the vector and the packaging constructs, the design of the SIV packaging cell line should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the SIV vector constructs that can be packaged. Copyright © 2002 John Wiley & Sons, Ltd.

     

High-level gene transfer to the cornea using electroporation

Background

Methods for gene transfer to the cornea that yield high‐level expression without inflammation or trauma are currently lacking. Because electroporation has proven effective for gene transfer in other tissues in terms of expression levels and safety, this study quantitatively evaluated its use in the cornea.

Methods

To evaluate the use of electroporation in the mouse cornea, plasmids expressing either luciferase or green fluorescent protein were injected intracorneally or subconjunctivally and square‐wave electric pulses were immediately applied to the eyes. Gene expression was quantified at later times and trauma and inflammation were monitored visually and by measuring interleukin‐6 (IL‐6) production.

Results

The application of electric pulses to eyes injected with plasmid resulted in nanogram levels of gene product expression. At an optimal field strength of 200 V/cm, no trauma, corneal edema or inflammation was observed. However, at higher field strengths, corneal damage was detected. Compared with injection of DNA alone, up to 1000‐fold more gene product was produced using electroporation. Expression was detected as early as 6 h post‐electroporation, remained high for 3 days, and decreased by 7 days. Gene expression was detected over the entire surface of the cornea in both epithelial and stromal layers.

Conclusions

These results demonstrate that electroporation is an excellent method for delivering genes to multiple cell layers within the mouse cornea and that it results in extremely high levels of gene expression with little, if any, inflammatory response or tissue damage, making this a very useful technique for corneal gene transfer. Copyright © 2001 John Wiley & Sons, Ltd.

     

Transfer of maternally administered fusogenic liposome-DNA complexes into monkey fetuses in a pregnancy model

Background

Materno‐fetal transfer of intravenously administered liposome‐plasmid DNA complexes has been demonstrated only in mice. Studies on its materno‐fetal transfer in the pregnant monkey model is needed because of critical differences in placental structure between primates including humans and rodents.

Methods

The reporter plasmid pEGFP‐C1 was formulated in cationic lipid containing polybrene and vesicular stomatitis virus G protein. The fusogenic liposome‐plasmid DNA complexes were intradermally injected into pregnant common marmosets (N=2), a New World monkey, near term. DNA extracted from fetal tissues was subjected to PCR for detection of the egfp gene. Confocal microscopy and immunostaining were performed to determine the sites of transgene expression in the fetal organs.

Results

The egfp gene was detected in fetal blood and major organs (heart, liver, lung). The encoded protein was mainly produced in the endothelial cells of blood vessels in the fetal lungs.

Conclusions

This is the first report on materno‐fetal transfer of intradermally administered fusogenic liposome‐plasmid DNA complexes and fetal expression of a transgene in primates. Copyright © 2002 John Wiley & Sons, Ltd.

     

Cationic lipids derived from glycine betaine promote efficient and non-toxic gene transfection in cultured hepatocytes

Background

The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non‐viral gene therapy.

Methods

A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12‐14‐16, 22‐24‐26) varying in their spacer and acyl chains.

Results

GB12, characterized by short [(CH₂)₁₀] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6^? and Lipofectin^?. We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12‐transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene.

Conclusions

The non‐toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy. Copyright © 2002 John Wiley & Sons, Ltd.

     

Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells

Background

The recently developed heterologous macrolide‐ (E.REX system) and streptogramin‐ (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines.

Methods

In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic‐dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter.

Results

These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression.

Conclusions

Due to their modular set‐up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell‐specific key regulation components. Copyright © 2002 John Wiley & Sons, Ltd.

     

Effect of chitosan‐alginate nanoparticles and ultrasound on the efficiency of gene transfection of human cancer cells

Background

Gene therapy has been used to treat a variety of health problems, but transfection inefficiency and the lack of safe vectors have limited clinical progress. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapy. The present study aimed to synthesize chitosan‐alginate nanoparticles that can be used as carriers of the pAcGFP1‐C1 plasmid and to use these nanoparticles with an ultrasound protocol to achieve high efficiency gene transfection.

Methods

Chitosan was complexed with alginate and the pAcGFP1‐C1 plasmid at different charge ratios to create chitosan‐alginate‐DNA nanoparticles (CADNs). The average particle size and loading efficiency were measured. Plasmid DNA retardation and integrity were analysed on 1% agarose gels. The effect of CADNs and ultrasound on the efficiency of transfection of cells and subcutaneous tumors was evaluated.

Results

In the CADNs, the average size of incorporated plasmid DNA was 600–650 nm and the loading efficiency was greater than 90%. On the basis of the results of the plasmid DNA protection test, CADNs could protect the transgene from DNase I degradation. The transgene product expression could be enhanced efficiently if cells or tumor tissues were first given CADNs and then treated with ultrasound.

Conclusions

The use of CADNs combined with an ultrasound regimen is a promising method for safe and effective gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.

     

Adeno-cosmid cloning vectors for regulated gene expression

Background

Adenovectors are widely used for efficient delivery of genes into a variety of cell types and organisms. However, the construction of the desired vector/genes combination, especially if it involves the cloning of several gene cassettes, can be laborious due to the large size of these vectors. New methods are needed to simplify the construction of complex combinations of gene cassettes into adenovectors.

Methods

Using simple cloning techniques and exploiting the λ‐phage packaging system, we devised efficient methods for the ‘selection’ of the desired vector constructs. Thus we generated a series of cosmids containing the adeno helper dependent (HD) backbone in which we inserted cis‐ and trans‐acting tetracycline (tet) elements for the regulation of any gene of interest. One of these cosmids has been used to produce an HD adenovirus carrying a tetracycline‐regulated gene expressing β‐galactosidase.

Results

We have demonstrated that the adeno‐cosmid system allows rapid and efficient cloning of genes of interest in helper dependent vectors, and described a prototype ‘ready‐to‐use’ vector in which any gene of interest can be easily expressed under the control of the tet system. The HD viruses produced with this novel methodology can be grown at high titers, can be easily separated from the helper adenovirus, and allow delivery and regulated gene expression in a variety of tissues.

Conclusions

Exploiting the λ‐packaging system, complex adeno constructs can be generated with a simple and reproducible protocol, which allows selection of the desired size construct, counterselecting for the frequently observed intramolecular recombinations and deletions. Copyright © 2002 John Wiley & Sons, Ltd.

     

Receptor-mediated delivery of an antisense gene to human brain cancer cells

Background

The goal of this work was the development of a gene targeting technology that will enable the delivery of therapeutic genes to brain cancer cells in vivo following intravenous administration. High‐grade brain gliomas overexpress the epidermal growth factor receptor (EGFR) and EGFR antisense gene therapy could reduce the growth of EGFR‐dependent gliomas.

Methods

A human EGFR antisense gene driven by the SV40 promoter in a non‐viral plasmid carrying elements that facilitate extra‐chromosomal replication was packaged in the interior of 85 nm pegylated immunoliposomes (PILs). The PILs were targeted to U87 human glioma cells with the 83‐14 murine monoclonal antibody (MAb) to the human insulin receptor (HIR).

Results

Confocal fluorescent microscopy demonstrated that the unconjugated HIR MAb is rapidly internalized by the glioma cells. Endocytosis followed by entry into the nucleus was also demonstrated for the HIR MAb conjugated PILs carrying fluorescein‐labeled plasmid DNA. The PILs delivered exogenous genes to virtually all cells in culture, based on β‐galactosidase histochemistry. The targeting of a luciferase gene to the U87 cells with the PILs resulted in luciferase levels in excess of 150 pg/mg protein after 72 h of incubation. The level of luciferase gene expression in the U87 cells achieved with the PIL gene targeting system was comparable to that with lipofectamine. Targeting the EGFR antisense gene to U87 glioma cells with the PILs resulted in more than 70% reduction in [³H]thymidine incorporation into the cells; this was paralleled by a 79% reduction in the level of immunoreactive EGFR.

Conclusion

The present work describes the targeting of an EGFR antisense gene to human brain cancer cells, which results in a 70–80% inhibition in cancer cell growth. PILs provide a new approach to gene targeting that is effective in vivo following intravenous administration without viral vectors. Copyright © 2002 John Wiley & Sons, Ltd.

     

Nonviral vector loaded collagen sponges for sustained gene delivery in vitro and in vivo

Background

Naked DNA and standard vectors have previously been used for gene delivery from implantable carrier matrices with great potential for gene therapeutic assistance of wound healing or tissue engineering. We have previously developed copolymer‐protected gene vectors which are inert towards opsonization. Here we examine their potency in carrier‐mediated gene delivery in comparison to standard vectors using a vector‐loaded collagen sponge model.

Methods

Equine collagen type I sponges were loaded by a lyophilization method with naked DNA, polyethylenimine (PEI)‐DNA, DOTAP/cholesterol‐DNA and copolymer‐protected PEI‐DNA. These preparations were characterized in terms of vector‐release, cell growth on the matrices and reporter gene expression by cells colonizing the sponges in vitro and in vivo. Subcutaneous implantation of sponges in rats served as an in vivo model.

Results

At the chosen low vector dose, the loading efficiency was at least 86%. Naked DNA‐loaded collagen matrices lost 77% of the DNA dose in an initial burst in aqueous buffer in vitro. The other preparations examined displayed a sustained vector release. There was no difference in cell growth and invasion of the sponges between vector‐loaded and untreated collagen grafts. Reporter gene expression from cells colonizing the sponges in vitro was observed for not more than 7 days with naked DNA, whereas the lipoplex and polyplex preparations yielded long‐term expression throughout the experimental period of up to 56 days. The highest expression levels were achieved with the PEI‐DNA‐PROCOP (protective copolymer) formulation. Upon subcutaneous implantation in rats, no luciferase expression was detected with naked DNA preparations. DOTAP/cholesterol‐DNA and PEI‐DNA‐loaded implants lead to reporter gene expression for at least 3 days, but with poor reproducibility. PEI‐DNA‐PROCOP collagen matrices yielded consistently the highest reporter gene expression levels for at least 7 days with good reproducibility.

Conclusions

With the preparation method chosen, lipoplex‐ and polyplex‐loaded collagen sponges are superior in mediating sustained gene delivery in vitro and local transfection in vivo as compared to naked DNA‐loaded sponges. Protective copolymers are particularly advantageous in promoting the tranfection capacity of polyplex‐loaded sponges upon subcutaneous implantation, likely due to their stabilizing and opsonization‐inhibiting properties. Copyright © 2002 John Wiley & Sons, Ltd.

     

Intratumoral activation of cyclophosphamide by retroviral transfer of the cytochrome P450 2B1 in a pancreatic tumor model. Combination with the HSVtk/GCV system

Background

Pancreatic cancer is one of the most aggressive human tumors and the development of new therapeutic approaches is particularly urgent since current therapies are not effective. The use of pro‐drug‐activating genes is a possible approach for cancer gene therapy.

Methods

The present study evaluated the efficiency of the cytochrome P4502B1 (CYP2B1) suicide gene that encodes the enzyme responsible for activating the pro‐drug cyclophosphamide (CPA), in pancreatic tumor cells invitro and in vivo. The effects on tumor growth of the combination of two suicide systems, CYP2B1/CPA and herpes simplex virus thymidine kinase gene/ganciclovir (HSVtk/GCV), were also studied.

Results

Retroviral CYP2B1 transfer followed by CPA treatment highly sensitized pancreatic tumor cells NP‐9, NP‐18, and NP‐31, and led to stabilization of tumor growth in a pancreatic tumor model. Differences in tumor volume at the end of the treatment were statistically significant when compared with animals injected with CPA alone. The combination of both suicide systems CYP2B1/CPA and HSVtk/GCV in vitro resulted in a potentiation of the killing effect. However, no potentiation was achieved in vivo, although retardation in tumor growth was evident.

Conclusions

The results show that in situ transduction of pancreatic tumor cells with the CYP2B1 gene by retroviral vectors clearly increases the sensitivity to CPA. Moreover, they suggest that in order to achieve a potentiation on cell killing when the two suicide systems HSVtk/GCV and CYP2B1/CPA are combined, co‐expression of both genes in the same tumor cell would be necessary. Copyright © 2002 John Wiley & Sons, Ltd.

     

Targeted gene therapy for rat glomerulonephritis using HVJ-immunoliposomes

Background

Kidney targeted gene transfer has been attempted by many researchers over the last 10 years; however, unfortunately, no reliable technique for gene transfer to the kidney has been established. At experimental level several in vivo gene transfer methods have been reported.

Methods

We were the first to report successful in vivo gene transfer into the kidney using the HVJ‐liposome method. Since then, this method has been modified to achieve highly efficient gene transfer. In this study, we have developed a renal glomerulus‐specific gene transfer method using HVJ‐liposomes with anti‐Thy 1 antibody, OX‐7.

Results

Following systemic delivery of fluoroisothiocyanate (FITC)‐labeled oligodeoxynucleotides (ODN) by HVJ‐liposomes coupled with OX‐7, we observed fluorescence in renal glomeruli from 2 h post‐administration. To examine the efficacy of this delivery system, NF‐κB or scrambled (SD) decoy ODN was administered by HVJ‐liposomes coupled with OX‐7 into a crescent glomerulonephritis, anti‐g lomerular b asement m embrane (GBM) model. Animals given SD decoy ODN developed severe glomerulonephritis by day 7 with heavy albuminuria, glomerular crescent formation and up‐regulated renal expression of IL‐1β and ICAM‐1. In contrast, NF‐κB decoy ODN treatment substantially inhibited the disease with a reduction in alubuminuria, histological damage and the renal expression of inflammatory cytokines.

Conclusions

This study has demonstrated that systemic delivery of HVJ‐liposomes coupled with OX‐7 results in efficient ODN transfer in rat glomeruli. NF‐κB, but not SD decoy ODN administered systemically via HVJ‐liposomes complexed with OX‐7 showed clear therapeutic potential for glomerulonephritis. This novel ODN transfer method combined with decoy strategy has the potential to lead to the establishment of a new therapeutic approach to glomerular diseases. Copyright © 2002 John Wiley & Sons, Ltd.