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.

     

Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses

Background

Helper‐dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high‐titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo.

Methods

Replication‐defective helper viruses were generated that are deleted for Ad E1, E2b and E3 genes, but which contain loxP sites flanking the packaging signal. Complementing Ad packaging cell lines (C7‐cre cells) were also generated by transfecting 293 cells with the Ad E2b genes encoding DNA polymerase and pre‐terminal protein, and with a cre‐recombinase plasmid.

Results

We show that C7‐cre cells allow efficient production of gutted Ad using ΔE1 + ΔE2b + ΔE3 helper viruses whose growth can be limited by cre‐loxP‐mediated excision of the packaging signal. Gutted Ad vectors carrying ～28 kb cassettes expressing full‐length dystrophin were prepared at high titers, similar to those obtained with E2b+ helpers, with a resulting helper contamination of <1%.

Conclusions

These new packaging cell lines and helper viruses offer several significant advantages for gutted Ad vector production. They allow gutted virus amplification using a reduced number of passages, which should reduce the chances of selecting rearranged products. Furthermore, the residual helper contamination in gutted vector preparations should be less able to elicit immunological reactions upon delivery to tissues, since E2b‐deleted vectors display a profound reduction in viral gene expression. Copyright © 2002 John Wiley & Sons, Ltd.

     

Genetic retargeting of adenovirus vectors: functionality of targeting ligands and their influence on virus viability

Background

We studied the ability of adenovirus type 5 (Ad5) to encapsidate new cellular ligands carried by their fibers to yield functional retargeted vectors for gene therapy. Recombinant Ad5 fibers containing shaft repeats 1 to 7 and an extrinsic trimerization motif, and terminated by its native knob or amino acid motifs containing RGD, have been rescued into infectious virions.

Methods

Polypeptide ligands of cell surface molecules, including single‐chain antibodies or epidermal growth factor, were cloned into recombinant fibers. Phenotypic analysis of fiber constructs and rescuing into the Ad5 genome were performed. Recombinant viruses were characterized with reference to fiber content, growth rate and infectivity.

Results

A major limiting factor for recovering viable recombinant Ad5 carrying fiber‐fused polypeptide ligands was apparently the ability of the ligand to fold correctly within the cellular cytoplasm. This constraint has previously not been systematically evaluated in the literature. Phenotypic analysis of the fiber‐ligand fusions showed that their degree of cytoplasmic solubility correlated with their ability to yield viable Ad5 vectors. Our results suggested that the fiber manipulations diminish virus growth rate, probably through different, opposing effects: (i) the reduced shaft length increases fiber solubility in the absence of the knob but (ii) diminishes virus entry, and (iii) the absence of the knob alters the overall protein composition of the virion and decreases its fiber copy number.

Conclusions

Based on our findings, cytoplasmic solubility and cytoplasmic ligand reactivity of fiber‐ligand fusion proteins are the best prediction criterion for viability and recovery of genetically retargeted Ad vectors. 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.

     

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.

     

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.

     

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.

     

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.

     

Cyanide bystander effect of the linamarase/linamarin killer-suicide gene therapy system

Background

The killer‐suicide system linamarase/linamarin (lis/lin) uses the plant gene linamarase (β‐glucosidase) to convert the cyanogenic glucoside substrate, linamarin, into glucose and cyanide. We have studied the bystander effect associated with this new system mediated by the production of the cyanide ion that diffuses freely across membranes.

Methods

Immunofluorescent staining of cells treated with an anti‐linamarase antibody allowed us to localize the enzyme within the cells. Flow cytometry was used to determine the sensitivity of different mixtures of cells, C6lis and C6gfp (green), to linamarin as a percentage of cell survival.

Results

We demonstrate here that rat glioblastoma C6 cells carrying the linamarase gene (lis), mixed with naive C6 cells and exposed to linamarin, induce generalized cell death. Cells expressing lis efficiently export linamarase, whereas linamarin enters cells poorly by endocytosis; as a result most of the cyanide is produced outside the cells. The study was facilitated by the presence of the green fluorescent protein (gfp) gene in the bystander population. As few as 10% C6lis‐positive cells are sufficient to eliminate the entire cell culture in 96 h.

Conclusions

This bystander mechanism does not preferentially kill toxic metabolite producer cells compared with bystander cells, thus allowing production of sufficient cyanide to cause tumor regression. In this report we confirm the potential of the lis/lin gene therapy system as a powerful tool to eliminate tumors in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector

Background

The pig lung, given its gross anatomical, histological and physiological similarities to the human lung, may be useful as a large animal model, in addition to rodents, in which to assess the potential of vectors for pulmonary airway gene transfer. The aim of this study was to assess the utility of the pig lung as a model of gene transfer to the human lung with a synthetic vector system.

Methods

The LID vector system consists of a complex of lipofectin (L), integrin‐binding peptide (I) and plasmid DNA (D). LID complexes containing a β‐galactosidase reporter gene under a CMV promoter or a control plasmid at1 mg/3 ml PBS, or 3 ml buffer, was administered to the right lower lobe ofthe pig lung through a bronchoscope. Pigs were culled at 48 h and lung sections prepared for immunohistochemical and histological analysis. Bronchoalveolar lavage fluid was collected and analysed for TNF‐α by ELISA.

Results

Immunohistochemical staining for the β‐galactosidase reporter gene indicated high efficiency of gene transfer by the LID vector to pig bronchial epithelium with 46% of large bronchi staining positively. There was no evidence for vector‐specific inflammation assessed by leukocytosis and cytokine production.

Conclusions

This study demonstrates the use of the pig for studies of gene transfer in the lung and confirms in a second species the potential of the LID vector for gene therapy of pulmonary diseases such as cystic fibrosis. 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.

     

Sustained delivery of therapeutic concentrations of human clotting factor IX--a comparison of adenoviral and AAV vectors administered in utero

Background

Prenatal somatic gene therapy has been considered for genetic disorders presenting with morbidity at birth. Haemophilia is associated with an increased risk of catastrophic perinatal bleeding complications such as intracranial haemorrhage, which could be prevented by gene transfer in utero. Prenatal gene therapy may be more promising than postnatal treatment, as the fetus may be more amenable to uptake and integration of therapeutic DNA and the immaturity of its immune system may permit life‐long immune tolerance of the transgenic protein, thus avoiding the dominant problem in haemophilia treatment, the formation of inhibitory antibodies.

Methods

Adenovirus serotype 5‐derived or AAV serotype 2‐derived vectors carrying human clotting factor IX (hfIX) cDNA or a reporter gene were administered intramuscularly, intraperitoneally or intravascularly to late‐gestation mouse fetuses. Both vector types were evaluated with respect to the kinetics of hfIX delivery to the systemic circulation and possible immune responses against the vector or the transgene product.

Results

Mice treated in utero by intramuscular injection of an adenoviral vector carrying hfIX cDNA exhibited high‐level gene expression at birth and therapeutic – albeit continuously decreasing – plasma concentrations of hfIX over the entire 6 months of the study. Adenoviral vector spread to multiple organs was detected by polymerase chain reaction (PCR). Intramuscular, intraperitoneal or intravascular application of AAV vectors carrying hfIX cDNA led to much lower plasma concentrations of hfIX shortly after birth, which appeared to decline during the first month of life but stabilized in some of the mice at detectable levels. No signs of immune responses were found, either against the different viral vectors or against hfIX.

Conclusion

This study demonstrates for the first time that sustained systemic delivery of a therapeutic protein can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy in utero and provides a basis for considering this concept as a preventive therapeutic strategy for haemophilia and perhaps also for other plasma protein deficiencies. Copyright © 2002 John Wiley & Sons, Ltd.