Joanne T. Douglas

RhoA and Cytoskeletal Disruption Mediate Reduced Osteoblastogenesis and Enhanced Adipogenesis of Human Mesenchymal Stem Cells in Modeled Microgravity

Spaceflight, aging, and disuse lead to reduced BMD. This study shows that overexpression of constitutively active RhoA restores actin cytoskeletal arrangement, enhances the osteoblastic phenotype, and suppresses the adipocytic phenotype of human mesenchymal stem cells cultured in modeled microgravity.

Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma

PurposeAlternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo.Experimental designHMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival.ResultsIntravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone.ConclusionInjected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

A system for the propagation of adenoviral vectors with genetically modified receptor specificities

The development of genetically modified adenovirus (Ad) vectors with specificity for a single cell type will require both the introduction of novel tropism determinants and the ablation of endogenous tropism. Consequently, it will not be possible to exploit the native cellular entry pathway in the propagation of these targeted Ad vectors. Based on the concept that Ad enters cells by a two-step process in which a primary receptor serves as a high affinity binding site for the Ad fiber knob, with subsequent internalization mediated by αv integrins, we designed two artificial primary receptors. The extracellular domain of one of these synthetic receptors was derived from a single-chain antibody (sFv) with specificity for Ad5 knob, while the second receptor consisted of an icosapeptide identified by biopanning a phage display library against Ad5 knob. Expression of either of these artificial virus-binding receptors in fiber receptor-negative cells possessing αv integrins conferred susceptibility to Ad infection. We then created a novel mechanism for cell binding by genetically modifying both the vector and the target cell. In this approach, six histidine (His) residues were incorporated at the C-terminal of the Ad fiber protein. The resultant Ad vector was able to infect nonpermissive cells displaying the cognate artificial receptor, containing an anti-His sFv. This strategy, comprising a genetically engineered Ad virion and a modified cell line, should be useful in the propagation of targeted Ad vectors that lack the ability to bind the native fiber receptor.

The therapeutic efficacy of adenoviral vectors for cancer gene therapy is limited by a low level of primary adenovirus receptors on tumour cells

Replication-defective adenoviral vectors are currently being employed as gene delivery vehicles for cancer gene therapy. To address the hypothesis that the therapeutic efficacy of adenoviral vectors is restricted by their inability to infect tumour cells expressing low levels of the primary cellular receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR), we have employed a pair of ovarian cancer cell lines differing only in the expression of a primary receptor for Ad5. This novel system thus allowed the direct evaluation of the relationship between the efficacy of an adenoviral vector and the primary receptor levels of the host cancer cell, without the confounding influence of other variable cellular factors. We demonstrate that a deficiency of the primary cellular receptor on the tumour cells restricts the efficacy of adenoviral vectors in two distinct cancer gene therapy approaches, TP53 gene replacement therapy and herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy. Moreover, we show that a deficiency of the primary receptor on the tumour cells limits the efficiency of adenovirus-mediated gene transfer in vivo. Since a number of studies have reported that primary cancer cells express only low levels of CAR, our results suggest that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realize the full potential of adenoviral vectors in the clinical setting.

Imaging and tissue biodistribution of 99mTc-labeled adenovirus knob (serotype 5)

Hepatic sequestration of systemically administered adenoviral vectors reduces the number of viral particles available for delivery to other tissues. The biological basis of this phenomenon was investigated using a new in vivo technique which permitted imaging in real time. Recombinant adenovirus serotype 5 knob (Ad5K) was radiolabeled with the gamma-emitter 99mTc (half-life = 6 h). Scatchard analysis of the 99mTc-Ad5K showed specific, high-affinity binding to U293 cells (Kd = 1.4 ± 0.5 nM), demonstrating that the radiolabeling process had no effect on receptor binding. In vivo dynamic imaging with an Anger gamma camera revealed that the liver binding followed an exponential rise to maximum, with a measured 100% extraction efficiency. Initially, the liver binding capacity was 3.1 ± 0.4 μg Ad5K, equivalent to approximately 17000 Ad5K molecules per liver cell. Liver binding was blocked by preincubation of Ad5K with neutralizing anti-Ad5K antibody; a 50% reduction in liver uptake was demonstrated by imaging. Unlabeled Ad5K was more effective in blocking liver uptake of 99mTc-Ad5K, whereas irrelevant unlabeled Ad3K had no effect. Imaging data for the liver uptake studies were in agreement with biodistribution determined by removing and measuring tissues. These data demonstrated that in vivo imaging is a sensitive tool for measuring changes to liver tropism. Similar imaging techniques can be applied to adenovirus vectors to measure specific targeting for gene therapy.

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.

The coxsackievirus and adenovirus receptor acts as a tumour suppressor in malignant glioma cells

The coxsackievirus and adenovirus receptor (CAR) is a membrane glycoprotein with a cytoplasmic domain, a transmembrane domain and an extracellular region consisting of two immunoglobulin-like domains, an amino-terminal immunoglobulin variable (IgV)-related domain (D1), which is distal to the cell surface, and a proximal IgC2 domain (D2). The coxsackievirus and adenovirus receptor has been shown to exhibit tumour suppression activity in human bladder and prostate cancer cells. In the current paper, we demonstrate that CAR is a tumour suppressor in glioma cells and that the extracellular D2 domain is not required for this inhibitory effect. This finding provides a biological basis for the observation that expression of CAR is downregulated in malignant glioma cells. This suggests that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realise the full potential of adenoviral vectors for cancer gene therapy.

Armed replicating adenoviruses for cancer virotherapy.

Conditionally replicating adenoviruses (CRAds) have many advantages as agents for cancer virotherapy and have been safely used in human clinical trials. However, replicating adenoviruses have been limited in their ability to eliminate tumors by oncolysis. Thus, the efficacy of these agents must be improved. To this end, CRAds have been engineered to express therapeutic transgenes that exert antitumor effects independent of direct viral oncolysis. These transgenes can be expressed under native gene control elements, in which case placement within the genome determines the expression profile, or they can be controlled by exogenous promoters. The therapeutic transgenes used to arm replicating adenoviruses can be broadly classified into three groups. There are those that mediate killing of the infected cell, those that modulate the tumor microenvironment and those with immunomodulatory functions. Overall, the studies to date in animal models have shown that arming a CRAd with a rationally chosen therapeutic transgene can improve its antitumor efficacy over that of an unarmed CRAd. However, a number of obstacles must be overcome before the full potential of armed CRAds can be realized in the human clinical context. Hence, strategies are being developed to permit intravenous delivery to disseminated cancer cells, overcome the immune response and enable in vivo monitoring of the biodistribution and activity of armed CRAds.

Retargeting of adenoviral infection to melanoma: combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA.

Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus‐mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single‐chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma‐associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti‐HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50‐fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367‐fold increase), CAR‐independent and HMWMAA‐mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild‐type fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54‐fold increased adenoviral gene transfer to CAR‐negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA‐targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma.

Targeting of Adenovirus via Genetic Modification of the Viral Capsid Combined with a Protein Bridge

ABSTRACT A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.