Natural Tropism Research Articles

Tumor-specific gene transfer via an adenoviral vector targeted to the pan-carcinoma antigen EpCAM.

The utility of adenoviral vectors for cancer therapy is limited due to their lack of specificity for tumor cells. In order to target adenovirus to tumor, the natural tropism of the adenovirus should be ablated and replaced by a tumor-specific binding domain. To this end, a neutralizing anti-fiber antibody conjugated to an anti-EpCAM antibody was created that targets the adenovirus to the EpCAM antigen present on tumor cells. The EpCAM antigen was chosen as the target because this antigen is highly expressed on a variety of adenocarcinomas of different origin such as breast, ovary, colon and lung, whereas EpCAM expression is limited in normal tissues. In these studies, the EpCAM-targeted adenovirus was shown to infect specifically cancer cell lines of different origin expressing EpCAM such as ovary, colon and head and neck. Gene transfer was blocked by excess anti-EpCAM antibody and dramatically reduced in EpCAM negative cell lines, thus showing the specificity of the EpCAM-targeted adenovirus. Importantly, infection with targeted adenovirus was independent of CAR, which is the natural receptor for adenovirus binding, since blocking of CAR with recombinant fiber knob did not affect infection with targeted adenovirus. Apart from the cancer cell lines, the efficacy of targeted viral infection was studied in freshly isolated primary human colon cancer cells. As colon cancer predominantly metastasizes to liver, and adenovirus has a high tropism for hepatocytes, we also sought to determine if the EpCAM-targeted adenovirus showed reduced infectivity of human liver cells. The bispecific antibody could successfully mediate gene transfer to primary human colon cancer cells, whereas it almost completely abolished infection of liver cells. This work thus demonstrates that EpCAM-targeted adenoviral vectors can be specifically directed to a wide variety of adenocarcinomas. This approach may prove to be useful for selective gene therapy of cancer.

Exploring brain circuitry with neurotropic viruses: new horizons in neuroanatomy.

There have been substantial advances in methods for defining connections among neurons over the past quarter century. However, most tracers have been limited in their ability to define populations of functionally related neurons that contribute to a multisynaptic circuit because they are not transported across synapses. As a result, the large body of literature that has employed these tracers has established regional associations between regions that must be further explored with electron microscopy and electrophysiological methods to define the synaptic relations among constituent neurons. Recently, neurotropic alpha herpesviruses have been used to visualize ensembles of neurons that contribute to polysynaptic networks. These pathogens invade permissive cells, replicate, and pass transynaptically to infect other neurons. In effect, the viruses become self-amplifying tracers whose natural tropism and invasiveness define populations of functionally related neurons. The recent increase in the use of this experimental approach has emerged from advances in our understanding of the life cycle of these viruses and the resulting evidence in support of specific transynaptic passage of progeny virus rather than infection by lytic release into the extracellular space. This article reviews the advances that have made this a viable experimental approach and considers ways in which this method has been creatively used to illuminate aspects of nervous system circuit organization that could not be defined with conventional tracers.

Strategies for achieving multiple layers of selectivity in gene therapy

Here we review the progress towards the development of targeted vectors for direct in vivo delivery in gene therapy. Currently, there are many separate approaches. These include: simple physical/anatomical localization of administration of the vector at the site where gene transfer is required; exploitation of natural tropisms of plasmid, viral and cellular vectors; and the use of molecular engineering to change the specificity of proteins and nucleic acids so that they specifically recognize target ligands expressed on/in the target cells. Unfortunately, each of these approaches is usually imperfect by itself. However, combinations of these strategies might produce vectors in which several layers of imperfect targeting give an overall level of specificity that can justify systemic delivery of vectors to treat human disease.

Use of a novel cross-linking method to modify adenovirus tropism.

Recombinant adenovirus (Ad) vectors can accomplish efficient in vivo gene transfer and thus are important in the context of a variety of gene therapy approaches. The cellular receptor for the Ad fiber knob is prevalent on a number of normal tissues which undermines the targeting of Ad to specific tumor cells. Therefore, the ablation of native Ad tropism and the introduction of novel Ad tropism are both necessary to target Ad vectors specifically to tumors. In this study, we have developed a flexible method for cross-linking the Fab fragment of a neutralizing anti-knob monoclonal antibody (1D6.14) to a cell receptor ligand. The cross-linking moieties are complementary low molecular weight recognition units, similar in concept to the avidin-biotin system. For proof of concept, we cross-linked 1D6.14 Fab to the basic fibroblast growth factor (FGF2). The Fab and FGF2 conjugates were synthesized and characterized both structurally and functionally. The conjugates were then complexed with an adenovirus vector carrying firefly luciferase (AdCMVLuc) and the resulting complex used to show infection of a number of tumor cell lines expressing FGF receptors. This cross-linking system should provide a rapid and convenient method of conjugating various ligands to the Fab fragment for targeting Ad vectors to different types of tumors.

Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.

Targeted adenoviral vectors for cancer gene therapy (Review)

In order to realise the full potential of gene therapy as a rational approach to the treatment of cancer, it will be necessary to achieve delivery of the therapeutic gene selectively to target tumour cells. Such cancer cell-specific gene delivery is mandated in the context of locoregional or compartmentalised carcinomas, and is also an absolute requirement for the treatment of disseminated disease. Moreover, underlying any cancer gene therapy approach is the need to achieve a high level of efficiency of gene transfer to the target cells. Of the existing viral and nonviral gene delivery vehicles, the adenoviral vector uniquely fulfils two requirements of an intravenously administered vector for cancer gene therapy: systemic stability and the ability to accomplish efficient transduction of cancer cells. However, it is necessary to modify native adenoviral tropism in order to achieve selective transduction of target tumour cells. A number of strategies have been developed for this purpose, involving genetic or immunological modifications to either of two adenoviral capsid proteins, the fibre and penton base. These strategies are designed to generate a targetable, injectable vector which would represent a major advance in the field of cancer gene therapy.

Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.

To expand the utility of recombinant adenovirus vectors for gene therapy applications, methods to alter native viral tropism to achieve cell-specific transduction would be beneficial. To this end, we are pursuing genetic methods to alter the cell recognition domain of the adenovirus fiber. To incorporate these modified fibers into mature virions, we have developed a method based on homologous DNA recombination between two plasmids. A fiber-deleted, propagation-defective rescue plasmid has been designed for recombination with a shuttle plasmid encoding a variant fiber gene. Recombination between the two plasmids results in the derivation of recombinant viruses containing the variant fiber gene. To establish the utility of this method, we constructed a recombinant adenovirus containing a fiber gene with a silent mutation. In addition, we generated an adenovirus vector containing chimeric fibers composed of the tail and shaft domains of adenovirus serotype 5 and the knob domain of serotype 3. This modification was shown to alter the receptor recognition profile of the virus containing the fiber chimera. Thus, this two-plasmid system allows for the generation of adenovirus vectors containing variant fibers. This method provides a rapid and facile means of generating fiber-modified recombinant adenoviruses. In addition, it should be possible to use this system in the development of adenovirus vectors with modified tropism to allow cell-specific targeting.

Tropism of sheep lentiviruses for monocytes: susceptibility to infection and virus gene expression increase during maturation of monocytes to macrophages

Visna lentiviruses have a natural tropism for cells of the macrophage lineage of sheep and goats, but virus replication in these cells in vivo is restricted so that only small quantities of virus are produced. One restricting factor suggested in previous studies is that virus replication is dependent on the maturity of the cells: the more mature the cell, the less restrictive the replication of the virus. Since monocytes in peripheral blood are precursors of macrophages, we investigated the effect of cell maturation on virus replication under limited control conditions in vitro by inoculating blood leukocytes with virus and retarding the maturation of monocytes to macrophages during cultivation in serum-free medium. Using enzyme markers that identified the cells in their resting monocytic stage (peroxidase) and mature macrophage stage (acid phosphatase) along with quantitative in situ hybridization and immunocytochemistry with viral reagents to trace the efficiency of virus replication, we correlated virus replication with cell maturation. Only a few monocytes were susceptible to infection, and virus replication did not extend beyond a low level of transcription of viral RNA. In the acid phosphatase-positive, maturing macrophage, susceptibility of the cells to infection was increased and virus replication was greatly amplified to the level of translation of viral polypeptides. However, virus maturation was delayed by 3 days until further cell maturation had occurred. Thus, the entire life cycle of the virus, from its attachment to the target cell to its maturation in the cell, was dependent on the level of maturation/differentiation of the monocytic cell.

Selective adherence as a determinant of the host tropisms of certain indigenous and pathogenic bacteria.

The relationship between the selective abilities of bacteria to adhere and their predilections for colonizing different mammalian hosts was investigated by using bacteria indigenous to the tongue dorsum of humans and rats as models. Streptococcus salivarius and S. sanguis averaged 22.6 and 2.8%, respectively, of the cultivable bacteria recovered from swab samples of the tonges of five humans, but these organisms were not indigenous on the tongues of rats (Charles River strain). S. faecalis and serum-requiring diphtheroids were consistently prominant on the tongues of rats, but they were not detected on the tongues of the humans examined. The ability of these organisms to adhere to the tongue surface of the hosts was compared by introducing mixtures of streptomycin-resistant strains into the mouths of human volunteers and rats. S. salivarius adhered in higher proportions to the dorsal tongue surface of humans than did strains of S. faecalis and the serum-requiring diphtheroid. S. sanguis also adhered to human tongues better than the serum-requiring diphtheroid. However, S. faecalis and the serum-requiring diphtheroid sorbed in higher proportions to the tongues of rats. In an in vivo assay, human strains of S. pyogenes and S. salivarius attached in higher numbers to buccal epithelial cells derived from humans than to those obtained from rats, whereas the reverse was observed with a serum-requiring diphtheroid derived from rats. Collectively, these studies show that bacteria sorb with a high degree of specificity to the tissues of different mammalian hosts, and the relative adherence of the organisms studied correlated with their natural host tropisms. The selective adherence of S. salvarius and S. faecalis was similar to the tongues of conventional and germ-free rats, suggesting that the presence of an indigenous bacterial flora did not significantly influence their attachment selectivity. Moreover, the ability of these organisms to colonize the tongues of gnotobiotic rats lacking an indigenous flora paralleled their adherence selectivity. Direct scanning microscopic observations indicated that the tongue dorsum of conventional rats is highly papillated but contains relatively sparse bacterial populations. Indigenous organisms colonized the bases of papillae on the anterior tip and lateral edges of the tongue as discrete microcolonies, but bacteria were rarely observed on other papillae. This localized and restricted pattern of colonization and the spatial distribution of the microcolonies of indigenous bacteria present also suggest that antagonistic interactions are unlikely to account for the bacterial tropisms observed for colonization of the tongues of rats.