Hidde J. Haisma

Endosomal escape pathways for delivery of biologicals

Despite continuous improvements in delivery systems, the development of methods for efficient and specific delivery of targeted therapeutic agents still remains an issue in biological treatments such as protein and gene therapy. The endocytic pathway is the major uptake mechanism of cells and any biological agents, such as DNA, siRNA and proteins. These agents become entrapped in endosomes and are degraded by specific enzymes in the lysosome. Thus, a limiting step in achieving an effective biological based therapy is to facilitate the endosomal escape and ensure cytosolic delivery of the therapeutics. Bacteria and viruses are pathogens which use different mechanisms to penetrate the membranes of their target cells and escape the endosomal pathway. Different mechanisms such as pore formation in the endosomal membrane, pH-buffering effect of protonable groups and fusion into the lipid bilayer of endosomes have been proposed to facilitate the endosomal escape. Several viral and bacterial proteins have been identified that are involved in this process. In addition, chemical agents and photochemical methods to rupture the endosomal membrane have been described. New synthetic biomimetic peptides and polymers with high efficacy in facilitating the endosomal escape, low pathogenicity and toxicity have been developed. Each strategy has different characteristics and challenges for designing the best agents and techniques to facilitate the endosomal escape are ongoing. In this review, several mechanisms and agents which are involved in endosomal escape are introduced.

Histone acetyl transferases as emerging drug targets.

Post-translational modifications, such as acetylation or phosphorylation, play a crucial role in the regulation of gene transcription in eukaryotes. Different subtypes of histone acetyl transferases (HATs) catalyze the acetylation of histones on specific lysine residues. A potential role of HATs in the pathology of cancer, asthma, COPD and viral infection has been described. This indicates that specific HAT inhibitors are potential tools for pharmacological research and might find therapeutic applications. This review focuses on the role of the HATs p300, CBP, PCAF and GCN5 in different diseases and the development of small-molecule inhibitors of these enzymes as potential drugs.

Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.

Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine‐conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)‐induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco‐conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol‐3 kinase pathway (PI3K) and nuclear factor‐κB were inhibited using LY 294002 and adenoviral overexpression of dominant‐negative IκB, respectively. The role of p38 and extracellular signal‐regulated protein kinase mitogen‐activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin‐D. Apoptosis was determined by measuring caspase‐3, ‐9, and ‐8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase‐8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCA‐induced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF‐κB activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA‐induced mitochondria‐controlled apoptosis by activating survival pathways. Supplemental material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/supplmat/index.html). (HEPATOLOGY 2004;39:1563–1573.)

Targeting of adenoviral vectors through a bispecific single-chain antibody

Recombinant adenoviral vectors are attractive in the context of cancer gene therapy because they are capable of delivering genes to a wide variety of tissues. The utility of adenoviruses is limited by their lack of specificity and by the absence of the receptor(s) for these viruses on many tumor cells. Redirecting adenoviral vectors to tissue- or tumor-specific targets can be achieved by using bispecific conjugates produced by chemical linkage of an anti-adenovirus antibody (Ab) and a ligand or Ab directed toward a specific target. To avoid the limitations of chemical conjugates, molecular conjugates of anti-fiber knob and ligand have been proposed. We present here a novel strategy that allows the production of recombinant bispecific single-chain Abs directed at cell surface molecules. A construct was made that encodes a neutralizing anti-adenovirus fiber single-chain Fv (scFv) Ab (S11) fused to a scFv Ab (425) directed against the epidermal growth factor receptor. The fusion protein markedly enhanced the infection efficiency of adenoviral vectors in epidermal growth factor receptor-expressing cell lines. The bispecific scFv could be purified and concentrated after binding of its 6His tag to a nickel column without significant loss of activity. This approach should permit the production of high quantities of active bispecific scFv for in vivo use. The universal design of the construct allows rapid screening for relevant specific scFv directed at cell surface antigens that can be incorporated into adenoviral targeting strategies.

A Novel Strategy to Modify Adenovirus Tropism and Enhance Transgene Delivery to Activated Vascular Endothelial Cells In Vitro and In Vivo

To assess the possibilities of retargeting adenovirus to activated endothelial cells, we conjugated bifunctional polyethylene glycol (PEG) onto the adenoviral capsid to inhibit the interaction between viral knob and coxsackie-adenovirus receptor (CAR). Subsequently, we introduced an alphav integrin-specific RGD peptide or E-selectin-specific antibody to the other functional group of the PEG molecule for the retargeting of the adenovirus to activated endothelial cells. In vitro studies showed that this approach resulted in the elimination of transgene transfer into CAR-positive cells, while at the same time specific transgene transfer to activated endothelial cells was achieved. PEGylated, retargeted adenovirus showed longer persistence in the blood circulation with area under plasma concentration-time curve (AUC) values increasing 12-fold compared to unmodified virus. Anti-E-selectin antibody-PEG-adenovirus selectively homed to inflamed skin in mice with a delayed-type hypersensitivity (DTH) inflammation, resulting in local expression of the reporter transgene luciferase. This is the first study showing the benefits of PEGylation on adenovirus behavior upon systemic administration. The approach described here can form the basis for further development of adenoviral gene therapy vectors with improved pharmacokinetics and increased efficiency and specificity of therapeutic gene transfer into endothelial cells in disease.

Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation

BACKGROUND/AIMS To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis. METHODS Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3. RESULTS Limited hepatocyte apoptosis and an increased expression of NF-kappaB-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-kappaB, NF-kappaB activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2. CONCLUSIONS GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-kappaB-regulated anti-apoptotic genes like A1 and cIAP2.

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.

A monoclonal antibody-beta-glucuronidase conjugate as activator of the prodrug epirubicin-glucuronide for specific treatment of cancer.

The anti-pan carcinoma monoclonal antibody (MAb) 323/A3, linked to E. coli-derived beta-glucuronidase (GUS) was used to study the tumour-site-selective activation of the prodrug Epirubicin-glucuronide (Epi-glu). Epi-glu was isolated from the urine of patients treated with Epirubicin (Epi) by reversed phase chromatography on a silica-C18 column. Epi-glu was stable in human blood and was not converted into Epi by A2780, MCF-7, or OVCAR-3 cancer cells, despite the presence of intracellular GUS. The stability of the prodrug was confirmed in BALB/c mice. MAb 323/A3 and GUS were linked through a stable thioether bond. The conjugate (1:1) was purified by ion exchange and gel filtration chromatography. Binding to target cells revealed an immunoreactivity of at least 60% and good retention of enzyme activity. A protein dye (sulforhodamine B) assay was used to analyse cytotoxicity. Epi (IC50 of 0.003-0.2 microM) was 100-1,000 times more toxic than Epi-glu (IC50 of greater than 20 microM), when cancer cells were exposed for 4 or 24 h to the drugs. The low cytotoxicity of Epi-glu was most likely due to the reduced cellular uptake rate of the prodrug (2.7 pmol 10(-6) cells min-1) as compared to that of the parent compound (25 pmol 10(-6) cells min-1). Pretreatment of antigen-positive cells with the 323/A3-GUS conjugate prior to prodrug exposure completely restored cytotoxicity as a result from hydrolysis of Epi-glu into Epi. Our results demonstrate that the 323/A3-GUS conjugate can specifically activate the stable non-toxic prodrug Epi-glu at the tumour cell level.

Recombinant adenovirus vectors with knobless fibers for targeted gene transfer

Adenoviral vector systems for gene therapy can be much improved by targeting vectors to specific cell types. This requires both the complete ablation of native adenovirus tropism and the introduction of a novel binding affinity in the viral capsid. We reasoned that these requirements could be fulfilled by deleting the entire knob domain of the adenovirus fiber protein and replacing it with two distinct moieties that provide a trimerization function for the knobless fiber and specific binding to the target cell, respectively. To test this concept, we constructed adenoviral vectors carrying knobless fibers comprising the α-helix trimerization domain from MoMuLV envelope glycoprotein. Two mimic targeting ligands, a Myc-epitope and a 6His-tag, were attached via a flexible linker peptide. The targeted knobless fiber molecules were properly expressed and imported into the nucleus of adenovirus packaging cells, where they were incorporated as functional trimers into the adenovirus capsid. Both ligands were exposed on the surface of the virion and were available for specific binding to their target molecules. Moreover, the knobless fibers mediated gene delivery into cells displaying receptors for the coupled ligand. Hence, these knobless fibers are prototype substrates for versatile addition of targeting ligands to generate truly targeted adenoviruses.

Efficient and Selective Gene Transfer into Primary Human Brain Tumors by Using Single-Chain Antibody-Targeted Adenoviral Vectors with Native Tropism Abolished

ABSTRACT The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduction and decreased toxicity to normal tissues. Targeting requires both the complete abolition of native tropism and the addition of a new specific binding ligand onto the viral capsid. Here we accomplished this by using doubly ablated adenoviral vectors, lacking coxsackievirus-adenovirus receptor and αv integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule. These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells. Targeted doubly ablated adenoviral vectors were also very efficient and specific with primary human tumor specimens. With primary glioma cell cultures, EGFR targeting augmented the median gene transfer efficiency of doubly ablated adenoviral vectors 123-fold. Moreover, EGFR-targeted doubly ablated vectors were selective for human brain tumors versus the surrounding normal brain tissue. They transduced organotypic glioma and meningioma spheroids with efficiencies similar to those of native adenoviral vectors, while exhibiting greater-than-10-fold-reduced background levels on normal brain explants from the same patients. As a result, EGFR-targeted doubly ablated adenoviral vectors had a 5- to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors. Hence, single-chain targeted doubly ablated adenoviral vectors are promising tools for cancer gene therapy. They should provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue.