Sonic hedgehog delivered by an adeno-associated virus protects dopaminergic neurones against 6-OHDA toxicity in the rat.

Direct intracerebral administration of sonic hedgehog (SHH) reduces 6-OHDA and MPTP toxicity to nigral dopaminergic cells in rats and primates. To determine whether transfection of the DNA sequence for SHH using viral vectors also protects against 6-OHDA toxicity, a type 2 adeno- associated virus (AAV) incorporating 600 base pairs of N-terminal SHH DNA was generated to induce SHH expression in rat striatum.AAV-SHH was injected into the striatum, 3 weeks prior to the initiation of an unilateral partial 6-OHDA nigro-striatal lesion. Animals receiving 4x10(7) viral particles of AAV-SHH showed a reduction in (+)-amphetamine induced ipsilateral turning over 4 weeks, when compared to animals receiving vehicle or a LacZ encoding vector. Following vehicle or AAV-LacZ administration, 6-OHDA caused a marked loss of striatal dopamine content and nigral tyrosine hydroxylase (TH) immunopositive cells. Following treatment with 4x10(7) viral particles of AAV-SHH the loss of striatal dopamine content was reduced and there was marked preservation of nigral dopaminergic cells. However, administration of 4x10(8) particles of AAV-SHH did not cause a significant change in (+)-amphetamine-induced rotation, striatal dopamine levels or the number of nigral TH immunoreactive cells following 6-OHDA lesioning compared to vehicle or AAV-LacZ treated animals. The results show that SHH delivered via a viral vector can protect dopaminergic neurons against 6-OHDA toxicity and suggest that this could be developed into a novel treatment for PD. However, the effects maybe dose limited due to uncoupling of hedgehog receptor signalling at higher levels of SHH expression.

Human and mouse IFN-beta gene therapy exhibits different anti-tumor mechanisms in mouse models.

Previously, we suggested that local human interferon-beta (IFN-beta) gene therapy with replication-defective adenoviral vectors can be an effective cancer treatment. Clinical trials to treat cancers with adenovirus expressing the human IFN-beta gene (IFNB1) has been planned. As a continued effort to explore the mechanisms of action of human IFN-beta gene therapy that can occur in the clinical setting, we tested mouse IFN-beta gene therapy in human xenograft tumors in both ex vivo and in vivo models. Delivery of the mouse IFN-beta gene (Ifnb) caused tumor inhibition; this effect was dependent on the indirect anti-tumor activities of IFN-beta, notably a stimulation of natural killer cells. IFN-beta does not show cross-species activity in its anti-proliferative effect and mouse IFN-beta does not cause as significant an anti-proliferative effect on mouse tumor cells as human IFN-beta causes on human tumor cells. Therefore, we believe that mouse models using either human IFN-beta or mouse IFN-beta gene transfer do not capture all aspects of the action of adenovirus-mediated human IFN-beta gene therapy that may be present in the clinical setting. Due to its multiple mechanisms of action, human IFN-beta gene therapy may be effective in treating human cancers that are either sensitive or resistant to the direct anti-proliferative effect of IFN-beta.

Eradication of intraperitoneal and distant tumor by adenovirus-mediated interferon-beta gene therapy is attributable to induction of systemic immunity.

Malignant mesothelioma remains an incurable disease for which immune-modulatory therapies, such as exogenous cytokines, have shown some promise. One such cytokine, IFN-beta, has potent antiproliferative and immunostimulatory activity in vitro, but its in vivo use has been limited by toxicity. We thus conducted studies evaluating intracavitary delivery of a replication-deficient adenoviral (Ad) vector encoding for the murine IFN-beta gene (Ad.muIFN-beta) in mouse models of malignant mesothelioma. In contrast to multiple injections of recombinant protein, a single i.p. injection of Ad.muIFN-beta into animals with established tumors elicited remarkable antitumor activity leading to long-term survival in >90% of animals bearing either AB12 or AC29 i.p. mesotheliomas. A control adenovirus vector had minimal antitumor effect in vivo. Significant therapeutic effects were also seen in animals treated with large tumor burdens. Importantly, treatment of i.p. tumor also led to reduction of growth in tumors established at a distant site (flank). A number of experiments suggested that these effects were attributable to an acquired CD8(+) T-cell-mediated response including: (a) the induction of long-lasting antitumor immunity; (b) loss of efficacy of Ad.muIFN-beta in tumor-bearing, immune-deficient (SCID, SCID/beige) mice; (c) detection of high levels of specific antitumor cytolytic activity from unstimulated splenocytes harvested from Ad.muIFN-beta-treated animals that was abolished by CD8(+) T-cell depletion; and (d) abrogation of antitumor effects of Ad.muIFN-beta in tumor-bearing CD8(+) T-cell-depleted animals. These data show that intracavitary IFN-beta gene therapy using an adenoviral vector provides strong CD8(+) T-cell-mediated antitumor effects in murine models of mesothelioma and suggest that this may be a promising strategy for the treatment of localized tumors such as mesothelioma or ovarian cancer in humans.

Systemic IFN-beta gene therapy results in long-term survival in mice with established colorectal liver metastases.

Most patients succumbing to colorectal cancer fail with liver-predominant metastases. To make a clinical impact in this disease, a systemic or whole-liver therapy may be required, whereas most cancer gene therapy approaches are limited in their ability to treat beyond local disease. As a preclinical model for cancer gene therapy, recombinant adenovirus containing the human IFN-beta (hIFN-beta) cDNA was delivered systemically in nude mouse xenograft models of human colorectal cancer liver metastases. The vector targeted hepatocytes that produced high levels of hIFN-beta in the liver, resulting in a profound apoptotic response in the tumors and significant tumor regression. hIFN-beta gene therapy not only resulted in improved survival and long-term cure in a micrometastatic model, but provided similar benefits in a clinically relevant gross disease model. A similar recombinant adenovirus containing the murine IFN-beta (mIFN-beta) cDNA also resulted in a therapeutic response and improved survival in syngeneic mouse models of colorectal cancer liver metastases. Depletion studies demonstrate a contribution of natural killer cells to this therapeutic response. The toxicity of an adenoviral vector expressing murine IFN-beta in a syngeneic model is also presented. These encouraging results warrant further investigation of the use of cancer gene therapy for targeting metastatic disease.

Sequestration of adenoviral vector by Kupffer cells leads to a nonlinear dose response of transduction in liver.

Systemic administration of a recombinant adenovirus encoding the human interferon-beta gene (H5.110CMVhIFN-beta) results in transduction of hepatocytes and detectable circulating levels of IFN-beta protein. In preclinical studies in mice, we noticed a distinctly nonlinear dose response, with low levels of virus (1-3 x 10(10) viral particles) yielding barely detectable levels of IFN-beta but with a higher viral dose (1 x 10(11) particles) resulting in disproportionately high IFN-beta levels. Further studies showed that transgene expression levels from low viral doses could be dramatically enhanced by coadministering an unrelated recombinant adenovirus (H5.110CMVlacZ), suggesting that there was a viral dose threshold effect for efficient viral transduction and/or IFN-beta expression. This enhancement of reporter expression by a nonreporter adenovirus, effective upon coadministration, was further enhanced by preadministration of H5.110CMVlacZ (up to 8 h), but was ineffective if the helper virus was administered as little as 5 min after the H5.110CMVhIFN-beta reporter virus. Our data suggest that the reticuloendothelial system plays a role in this threshold effect, such that low doses of virus are efficiently taken up by the RES/Kupffer cells without leading to appreciable transgene expression, whereas high doses saturate these cells and are able to productively transduce hepatocytes. A better understanding of this phenomenon could have an impact on gene therapy clinical trial safety and efficacy.

Baculovirus vector requires electrostatic interactions including heparan sulfate for efficient gene transfer in mammalian cells.

BACKGROUND: Recently, several reports have described the ability of recombinant baculoviruses to transduce a variety of mammalian cells. Yet, mechanisms involved in baculovirus entry in those cells remain largely unexplored, particularly at the primary binding step of the virions to the cell membrane. METHODS: This report focused on the primary virus-cell interactions that lead to in vitro transduction of human 293 cells using a polyhedrin-deleted baculovirus harboring a CMV-driven beta-galactosidase gene (BacLacZ). RESULTS: Infection rate monitored for 8 h and transduction rate with a multiplicity of infection of up to 800 were, both, non-saturable. Temperatures from 37 degrees C to 4 degrees C dramatically impaired BacLacZ but not adenovirus cell attachment. Competitive infections performed with an excess of a non LacZ-expressing baculovirus hardly competed at a 1/1 ratio. Consistent with an adsorptive binding process onto the cell surface, interactions through electrostatic charges between both viral and cell membranes appeared to be critical for BacLacZ transduction. The addition of polybrene to the cells prior to or during the infection prevented both virus binding and LacZ gene transfer, suggesting the involvement of negatively charged epitopes exposed at the cell surface. The simultaneous presence of the highly charged heparin abrogated BacLacZ binding to the cell surface and subsequent gene transfer. Lastly, direct in vitro binding of BacLacZ to heparin but not BSA columns could be demonstrated after elution of infectious BacLacZ virus in high salt molarity. CONCLUSION: Electrostatic charges play a critical role during the first step in mammalian cell transduction mediated by a recombinant baculovirus.

Interferon-beta gene therapy inhibits tumor formation and causes regression of established tumors in immune-deficient mice.

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-beta has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-beta concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-beta gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-beta gene delivery into established tumors generated high local concentrations of IFN-beta, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-beta gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein.

Baculovirus vectors recently have been shown to be capable of efficient transduction of human hepatoma cells and primary hepatocytes in culture. This paper describes the generation of a novel recombinant baculovirus (VGZ3) in which the vesicular stomatitis virus glycoprotein G (VSV G) is present in the viral envelope. The gene encoding VSV G was inserted into the baculovirus genome under the control of the polyhedrin promoter such that it was expressed at very high levels in infected insect cells but not in mammalian cells. Expression of the lacZ reporter gene was driven by a promoter that is functional in mammalian cells (the Rous sarcoma virus long terminal repeat). We show by Western analysis that VSV G protein was present in purified baculovirus preparations. A VSV G monoclonal antibody blocked transduction of mammalian cells by VGZ3. This virus was morphologically distinct from baculovirus lacking VSV G, with virions adopting an oval rather than rod-shaped morphology. VGZ3 transduced human hepatoma cells in vitro at an efficiency roughly 10-fold greater than baculovirus lacking VSV G (the virus Z4). VGZ3 was also capable of transducing cell lines that could not be transduced efficiently by Z4. We provide evidence that VSV G protein may enhance transduction by increasing the efficiency of escape of baculovirus from intracellular vesicles rather than by increasing cell binding or uptake of the virus. The possible use of this and related baculoviruses in gene therapy is discussed.

Interferon-beta induces S phase accumulation selectively in human transformed cells.

Interferons (IFNs) generally have been characterized as antiproliferative cytokines. The cell cycle arrest in G1/G0 phase induced by type I IFNs, especially IFN-alpha, was recognized as a manifestation of their antiproliferative effects. In this article, we report that the cell cycle block in G1/G0 is observed mainly in certain cell types, such as Daudi Burkitt's lymphoma cells. In a variety of human transformed cells, but not nontransformed primary cells, IFN-beta and IFN-alpha induced a significant increase in the S phase population. The increase appeared to be due to a continued S phase entry and subsequently a failure of S phase cells to transit efficiently into G2 and M phases. The ability of tumor cells to exhibit the S phase effect correlated with proper IFN signaling and loss or inactivation of the normal G1 checkpoint conferred by the retinoblastoma protein (pRB). Overriding the G1 checkpoint switched human nontransformed primary cells from nonresponsive to sensitive to the IFN-induced effect. Therefore, the cell cycle regulatory machinery could function, at least in part, as a determining factor that affects the IFN-induced cell cycle effect. The IFN effect in transformed cells may suggest intriguing prospects for combinatorial therapies for cancer.

Differential cell cycle effects induced by E2F1 mutants.

Expression of two types of transactivation-defective E2F1 mutants in human Rb-/- tumor cells led to an increase in the proportion of cells in the G1 phase of the cell cycle as determined by FACS analysis. Experiments revealed two different mechanisms of action. One mutant type induced a G1 arrest after the restriction point, with cells phenotypically at a cell cycle stage later than G1. The action of this mutant was, at least in part, dependent on specific DNA binding and was over-ridden by co-expression of its wild-type counterpart. The other mutant type, which is defective in DNA binding, slowed the G1 progression and restored a checkpoint for cell cycle withdrawal. The G1 phase withdrawal of these tumor cells allowed the initiation of skeletal muscle cell differentiation. Thus, E2F1 appears to have two different functions before and after the cell cycle restriction point. This report also may provide a basis for a gene therapy approach for certain human cancers.

Sequences flanking the core DNA-binding domain of bovine papillomavirus type 1 E2 contribute to DNA-binding function.

We have compared a series of molecular constructs that contain the minimal DNA-binding and dimerization domain of bovine papillomavirus type 1 (BPV-1) E2 alone or this binding domain plus the adjacent 16 or 40 amino acids to test the role of the flanking sequences in E2 function. The presence of these sequences resulted in an up to eightfold increase in the affinity of E2 for its target DNA and stabilized the protein against denaturation both in the absence of DNA and in the form of DNA-protein complexes. In addition, an aspartic acid-to-tyrosine mutation within the flanking region blocked DNA binding and function. These data demonstrate that sequences flanking the core domain contribute to E2 function and are, in fact, an integral part of the DNA-binding domain of BPV-1 E2.

Tat-mediated protein delivery can facilitate MHC class I presentation of antigens.

We have previously shown that the tat protein of HIV-1 can be used as a carrier to promote the intracellular delivery of heterologous proteins. Here we have tested if the tat-delivery technology can be used to direct MHC class I presentation of native protein, using ovalbumin (OVA) as a model system. We show that a tat-ovalbumin conjugate (tatOVA) can be delivered into cells and that subsequent processing and presentation occurs, resulting in effective and specific killing of these target cells by an OVA specific cytotoxic T-lymphocyte (CTL) line. Comparison with the E.G7 line that expresses the OVA gene indicates that tat-mediated delivery is as efficient as endogenous expression in this system. Tat-mediated antigenic protein delivery may be useful both as a research technique and, potentially, as a therapeutic or prophylactic vaccine.

The BPV-1 E2 DNA-contact helix cysteine is required for transcriptional activation but not replication in mammalian cells.

The papillomavirus E2 protein contains an amino-terminal region thought necessary and sufficient to support transcriptional activation and a carboxy-terminal region shown to direct sequence-specific DNA binding and dimerization. A cysteine residue in the center of the E2 DNA recognition helix is highly conserved among papillomavirus E2 proteins. Mutations of this cysteine in bovine papillomavirus type 1 E2 to serine and glycine resulted in proteins which failed to activate E2-dependent promoters in mammalian cells. These E2 mutants were DNA-binding competent, dimeric, and nuclear. When fused to the VP16 transactivation domain, C-terminal regions of E2 containing the mutations at 340 supported transcriptional activation, indicating that the heterologous trans-activation domain did not require cysteine in the DNA-binding helix as did the full-length E2 transactivating protein. Although cysteine-340 was required for transcriptional activation it was not required for DNA replication in vivo. Together, these results suggest that the E2 DNA-binding domain may directly contribute to functions of transcriptional activation previously thought limited to the N-terminal domain.

Endosomolytic activity of cationic liposomes enhances the delivery of human immunodeficiency virus-1 trans-activator protein (TAT) to mammalian cells.

We have explored the use of cationic liposomes to deliver the human immunodeficiency virus-1 trans-activator protein tat using a reporter gene expression assay. The human epidermoid carcinoma cell A431 stably transfected with a reporter gene under the control of human immunodeficiency virus-1 promoter was used as a target cell. Phosphatidylcholine-containing cationic liposomes had no detectable tat delivery activity. In contrast, delivery of tat was enhanced by up to 150-fold using cationic liposomes enriched with dioleoyl phosphatidylethanolamine (DOPE), a lipid which readily transforms a bilayer into a nonbilayer structure. Enhanced delivery of tat by DOPE-containing liposomes was most likely the result of the endosomolytic activity of the liposome. This phospholipid-rich formulation showed no toxicity at concentrations sufficient for maximal delivery of tat. A variety of cationic liposome formulations which contain DOPE were tested successfully for tat delivery.

Increased cellular uptake of the human immunodeficiency virus-1 Tat protein after modification with biotin.

The human immunodeficiency virus-1 Tat protein can efficiently enter cells when added exogenously in tissue culture. Using the transactivation activity of Tat as a measure of intracellular delivery, we found that the addition of hydrophobic groups to Tat potentiated its uptake. Biotin was the most promising of the reagents tested and we characterized this effect in more detail. When coupled through a cysteine thiol, the addition of a single biotin to Tat increased activity by about six-fold. Increased activity was only seen with reducible biotin analogs, as modification with noncleavable analogs is known to block Tat transactivation activity. Biotin had no effect on Tat uptake when mixed with Tat without cross-linking. Recently, Tat was used as a carrier to direct the uptake of heterologous proteins into cells. We have used RNase as a model system for studying Tat-mediated uptake and found that biotin also increased the delivery of a Tat37-58-RNase conjugate. The increased uptake of Tat and Tat conjugates by addition of hydrophobic groups may significantly enhance the usefulness of Tat as a delivery vehicle, and the approach may be applicable to other systems.

Codelivery to mammalian cells of a transcriptional factor with cis-acting element using cationic liposomes.

The human immunodeficiency virus-1 transactivator protein (tat) was codelivered efficiently with a reporter gene under the control of a tat-responsive DNA element using different formulations of cationic liposomes. Expression of a tat-responsive reporter gene was induced by incubating cells with a mixture of purified recombinant tat protein, reporter DNA, and liposomes. Different cell lines were tested successfully as targets for the codelivery. Tat was shown to trans-activate the codelivered virus promoter specifically in the cells tested. Codelivery of tat with DNA is a useful model for studying the function of trans-acting factors and their cis-acting DNA elements. The currently available methods such as foot-printing only reveal the binding, but not the functional consequence of the binding, of the factor with the element. In addition, this system may prove useful as a model for high level and regulated transgene expression in target cells.