Coxsackie and Adenovirus Receptor Binding Ablation Reduces Adenovirus Liver Tropism and Toxicity

Abstract

Human adenovirus-based vectors have emerged as a new promising vehicle for in vivo gene transfer-mediated therapy. However, the full potential of this methodology has not been fully realized because of the nonspecific tissue distribution of adenoviral vectors. Adenovirus infection is initiated by forming a complex between the fiber protein and a ubiquitously expressed host cell membrane protein, coxsackie B virus and adenovirus receptor (CAR). Therefore, ablating the adenovirus vector's ability to bind to the CAR is the first step in redirecting adenoviral tropism. To ablate CAR binding, we mutated the Bbeta sheet of the fiber knob, generating CAR-binding ablated replication-incompetent (dl-K420A-Z) and replication-competent (YKLK420A) adenoviral vectors. The in vitro transduction efficiency of dl-K420A-Z was significantly reduced in comparison to dl-LacZ carrying the wild-type fiber in CAR-positive cells but not in CAR-negative cells, suggesting that the mutation introduced in the Bbeta sheet of the fiber knob could disable the CAR-dependent transduction pathway. The in vivo transduction was also dramatically reduced in the liver and other organs for mice treated with dl-K420A-Z, compared with a cognate control vector, dl-LacZ. Concomitant with this attenuated gene transfer efficiency in vivo was a substantial reduction in the amount of general toxicity observed in the YKL-K420A-treated mice. Diminished toxicity was surmised from quantitative measurement of serum level of enzymes for liver and kidney function, hematologic chemistries, histopathology, and differences in lethality. Significant decrease in serum transaminases (alanine transferase [ALT] and aspartate transferase [AST]) was observed in mice treated with YKL-K420A. In addition, the lethality was lower in the YKLK420A- treated groups compared to the YKL-1-treated groups at all doses examined. Furthermore, the hepatopathologic analysis revealed that YKL-1 induced focal zonal necrosis and hepatocyte degeneration, while YKL-K420A induced mild spotty necrosis. In summary, this decreased vector tropism of CAR-binding ablated adenoviruses in normal tissues may increase the amount of virus available for infecting tumor cells and thus increase the antitumor efficacy with fewer unwanted side effects.

Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes

“…This has been driven by conflicting data relating to the loss of liver-targeting capacity of adenovirus type 5 vectors mutated in their ability to bind CAR. [4][5][6] As such, vectors with mutations that ablate CAR binding fail to retarget efficiently to alternate sites systemically when targeting peptides are inserted in the fiber (reviewed by Nicklin et al 7 ) even though retargeting in vitro or locally to defined tissues is efficacious. 8,9 A recent report described the importance of circulating factors (FIX and complement factor C4BP) in supporting adenovirus type 5 and type 35 delivery to the liver.…”

Coagulation Factors IX and X Enhance Binding and Infection of Adenovirus Types 5 and 31 in Human Epithelial Cells

“…These efforts have attempted to disrupt binding to CAR, integrins, and/or heparan sulfate (8,22,23,25,28,39,40,52); however, the results from these experiments have been inconsistent. Transduction of hepatocytes by species C-based vectors was recently suggested to depend on the binding of the most abundant structural protein of the virus particle, the hexon, to coagulation factor X (FX); the latter mediates indi-rect viral binding to cell surface heparan sulfate proteoglycans on hepatocytes (21,42).…”

Dendritic Cell-Based Genetic Immunotherapy for Ovarian Cancer

“…Studies by Vigne et al, [27] suggest that simultaneous ablation of CAR and αv-integrin binding, by introducing mutations in the fiber knob domain and deleting the RGD motif in the penton base respectively, fails to reduce Ad5 liver tropism. While studies by Kim JH et al, [28] suggest that ablation of CAR binding, by introducing mutation in the β sheet of the fiber knob, reduces adenovirus liver tropism and toxicity. Our results supports the results of Kim JH et al, and suggests the fiber-knob domain of the adenoviral particle does play an important role in infecting liver cells, as it is this domain that is masked by the CFm40L adapter molecule in the CD40-targeted adenoviral vector.…”

“…This approach has shown therapeutic effects in weakly immunogenic tumors in experimental models. In the same regard, DCs have been pulsed with total soluble antigens from tumor extracts and shown to elicit tumor-specific immunity [28,41,42]. Although both eluted tumor peptides and tumor lysates do represent individualized approaches for each patient's tumor, the fact that they rely on the availability of tumor material restricts their use.…”

“…Furthermore, the delivery of genetic material allows for prolonged expression and therefore presentation of antigens. Both nonviral [21,26,[43][44][45] and viral [22,25,28,33,35,42,46-53] methods have been described for introduction of DNA sequences, which have shown to promote specific CTL responses and protective and therapeutic anti-tumor immunity of tumors expressing the antigen sequence delivered. However, because the endogenously expressed antigens may have only limited access to the class II presentation pathway, it is possible that there will be a deficiency in generating CD4+ T helper cells, required for an effective and durable CTL response.…”