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.

     

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.

     

Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate-mediated targeting in vitro and prolonged plasma circulation in vivo

Background

Gene delivery vectors based on poly(L ‐lysine) and DNA (pLL/DNA complexes) have limited use for targeted systemic application in vivo since they bind cells and proteins non‐specifically. In this study we have attempted to form folate‐targeted vectors with extended systemic circulation by surface modification of pLL/DNA complexes with hydrophilic polymers.

Methods

pLL/DNA complexes were stabilised by surface modification with a multivalent reactive polymer based on alternating segments of poly(ethylene glycol) and tripeptides bearing reactive ester groups. Folate moieties were incorporated into the vectors either by direct attachment of folate to the polymer or via intermediate poly(ethylene glycol) spacers of 800 and 3400 Da.

Results

Polymer‐coated complexes show similar morphology to uncoated complexes, their zeta potential is decreased towards zero, serum protein binding is inhibited and aqueous solubility is substantially increased. Intravenous (i.v.) administration to mice of coated complexes produced extended systemic circulation, with up to 2000‐fold more DNA measured in the bloodstream after 30 min compared with simple pLL/DNA complexes. In further contrast to simple pLL/DNA complexes, coated complexes do not bind blood cells in vivo. Folate receptor targeting is shown to mediate targeted association with HeLa cells in vitro, leading to increased transgene expression. We demonstrate for the first time that DNA uptake via the folate receptor is dependent on pEG spacer length, with the transgene expression relatively independent of the level of internalised DNA.

Conclusions

We show increased systemic circulation, decreased blood cell and protein binding, and folate‐targeted transgene expression using pLL/DNA complexes surface‐modified with a novel multireactive hydrophilic polymer. This work provides the basis for the development of plasma‐circulating targeted vectors for in vivo applications. Copyright © 2002 John Wiley & Sons, Ltd.

     

Subretinal delivery of adeno‐associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice

Background

Adeno‐associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long‐term therapy is the development of immune responses against the vector or transgene product.

Methods

We evaluated cellular and humoural responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression.

Results

Following subretinal administration of vector, splenocytes and T‐cells from draining lymph nodes showed minimal activation following stimulation by co‐culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low‐dose AAV.hRPE65.hRPE65 to both eyes of RPE65^?/? mice resulted in transgene expression and functional rescue, but re‐administration of high‐dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye.

Conclusions

These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose‐dependent. Low‐dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration. Copyright © 2009 John Wiley & Sons, Ltd.

     

Inhibition of HBV gene expression and replication by stably expressed interferon-alpha1 via adeno-associated viral vectors

Background

Interferon‐α2 (IFNα2) is routinely used for anti‐hepatitis B virus (HBV) treatment. However, the therapeutic efficiency is unsatisfactory, particularly in East Asia. Such inefficiency might be a result of the short half‐life, relatively low local concentration and strong side‐effects of interferons. Frequent and repeated injection is also a big burden for patients. In the present study, a single dose of vector‐delivered IFNα1 was tested for its anti‐HBV effects.

Methods

Adeno‐associated viral vector (AAV‐IFNα1) was generated to deliver the IFNα1 gene into hepatocytes. IFNα1, hepatitis B surface (HBsAg) and e (HBeAg) antigens were measured by enzyme‐linked immunosorbent assay and/or western blotting. The level of viral DNA was measured by quantitative real‐time polymerase chain reaction.

Results

AAV‐IFNα1 effectively transduced HBV‐producing cells (HepAD38) and mouse hepatocytes, where IFNα1 was expressed in a stable manner. Both intracellular and extracellular HBsAg and HBeAg were significantly reduced in vitro. In the HBV‐producing mice, the concentration of IFNα1 in the liver was eight‐fold higher than that in plasma. Compared with control groups, HBeAg/HBsAg antigen levels were reduced by more than ten‐fold from day 1–5, and dropped to an undetectable level on day 9 in the AAV‐IFNα1 group. Concurrently, the level of viral DNA decreased over 30‐fold for several weeks.

Conclusions

A single dose administration of AAV‐IFNα1 viral vector displayed prolonged transgene expression and superior antiviral effects both in vitro and in vivo. Therefore, the use of AAV‐IFNα1 might be a potential alternative strategy for anti‐HBV therapy. Copyright © 2008 John Wiley & Sons, Ltd.

     

Preparation of dry powder dispersions for non-viral gene delivery by freeze-drying and spray-drying

Background

Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze‐drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non‐viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze‐drying and spray‐drying, a one‐step process for producing dry and respirable powders, as methods for preparing non‐viral respiratory gene delivery systems.

Methods

Lipid:polycation:pDNA (LPD) vectors comprising 1,2‐dioleoyl‐3‐trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP‐N1 in 3% lactose solution were either snap‐frozen and lyophilised or spray‐dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence.

Results

Lyophilisation produced large, irregularly shaped particles prior to (mean diameter ～21 µm) and following (mean diameter ～18 µm) coarse grinding. Spray‐drying produced uniformly shaped spherical particles (mean diameter ～4 µm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray‐dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage.

Conclusions

Spray‐drying has utility for producing stable, efficient and potentially respirable non‐viral dry powder systems for respiratory gene delivery. Copyright © 2002 John Wiley & Sons, Ltd.

     

Transfectant mosaic spheroids: a new model for evaluation of tumour cell killing in targeted radiotherapy and experimental gene therapy

Background

We describe an in vitro tumour model for targeted radiotherapy and gene therapy that incorporates cell population heterogeneity.

Materials and methods

Transfectant mosaic spheroids (TMS) and transfected mosaic monolayers (TMM) are composed of two cell populations derived from a single cell line. The cells of one population were transfected with the noradrenaline transporter gene (NAT), allowing active uptake of a radiolabelled targeting agent meta‐[¹³¹I]iodobenzylguanidine ([¹³¹I]MIBG); the other population of cells was derived from the same parent line and transfected with a marker gene – green fluorescent protein (GFP). After treatment with [¹³¹I]MIBG, cell kill was determined in TMM by clonogenic assay and in TMS by clonogenic assay and spheroid growth delay.

Results

We have used the TMS model to assess the ‘radiological bystander effect’ (radiation cross‐fire) conferred by the β‐emitting radiopharmaceutical [¹³¹I] MIBG whose cellular uptake is facilitated by the transfected gene encoding NAT. We show that cell killing by [¹³¹I]MIBG in both TMS and TMM cultures increased in direct proportion to the fraction of NAT‐transfected cells and that the degree of cell killing against fraction transfected was greater in TMS, suggestive of a greater bystander effect in the three‐dimensional culture system.

Conclusions

TMS provide a useful model for assessment of the effectiveness of targeted radiotherapy in combination with gene therapy when less than 100% of the target cell population is expressing the NAT transgene. Further, this novel model offers the unique opportunity to investigate radiation‐induced bystander effects and their contribution to cell cytotoxicity in radiotherapy and other gene therapy applications. Copyright © 2002 John Wiley & Sons, Ltd.

     

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.

     

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.

     

Production of helper-dependent adenovirus vector relies on helper virus structure and complementing

Background

The helper‐dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD‐Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD‐Ad vector is generally lower than that of an E1‐deleted first‐generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD‐Ad vector.

Methods

To study this question and improve HD‐Ad vector production, we have generated four different helper viruses with a wild‐type or deleted E3 region, and with a relocated loxP. We have also constructed a first‐generation vector with a wild‐type E3 region and without the loxP site. We compared the replication of these viruses in Cre‐positive and ‐negative cells and studied their complementing for HD‐Ad vector production.

Results

Viruses with deleted E3 formed smaller plaques and produced lower titer compared with viruses containing the E3 region. The site where a loxP is inserted can also affect virus replication. Higher yield of HD‐Ad vector was obtained when a helper virus with wild‐type E3 was used. We also showed that deletion of the packaging signal in a helper virus through loxP/Cre interaction decreased the viral DNA complementing ability.

Conclusions

Although the E3 region is not essential for adenovirus replication in vivo, deletion of this region attenuates virus replication. Production of HD‐Ad vector can be further improved by modifications in helper virus structure. 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.

     

Phenotypic rescue after adeno-associated virus-mediated delivery of 4-sulfatase to the retinal pigment epithelium of feline mucopolysaccharidosis VI

Background

Mucopolysaccharidosis VI (MPS VI), due to recessively inherited 4‐sulfatase (4S) deficiency, results in lysosomal storage of dermatan sulfate in numerous tissues. Retinal involvement is limited to the retinal pigment epithelium (RPE). This study aimed to determine whether recombinant adeno‐associated virus (AAV)‐mediated delivery of 4S would reverse the RPE pathology seen in MPS VI cats.

Methods

AAV.f4S, containing the feline 4S cDNA, was delivered unilaterally to eyes of affected cats by subretinal or intravitreal injection. Contralateral eyes received AAV with the green fluorescent protein (GFP) reporter gene as control. At 2–11 months post‐injection, the cats were sacrificed and the treatment effects were evaluated histologically.

Results

By ophthalmoscopy and histological analyses, GFP was evident as early as 4 weeks and persisted through the latest time point (11 months). Untreated and AAV.GFP‐treated diseased retinas contained massively hypertrophied RPE cells secondary to accumulation of dilated lysosomal inclusions containing dermatan sulfate. MPS VI eyes treated subretinally with AAV.f4S had minimal RPE cell inclusions and, consequently, were not hypertrophied.

Conclusions

AAV‐mediated subretinal delivery of f4S provided correction of the disease phenotype in RPE cells of feline MPS VI, supporting the utility of AAV as a vector for the treatment of RPE‐specific as well as lysosomal storage diseases. 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.

     

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.