Fiber-modified Vectors Research Articles

47. A Model To Determine the In Vivo Fate of Defined Adenovirus Immune Complexes

Vectors based on Adenovirus type 5 (Ad5) are well suited for the purpose of genetic vaccination due to their high immunogenicity. However, it is known that immune complexes formed upon contact with vector-specific antibodies (Abs) can impede gene transfer and influence vector immune profiles. To model, analyze, and understand the effects of immune complex formation on in vivo biodistribution and vector immunogenicity, we established a novel model based on chemical tagging of Ad capsomers with a carbohydrate epitope. Of note, this model allows to direct antibodies to specific capsid regions and thus to generate highly defined Ad immune complexes independent of species and MHC restrictions.As an artificial immunogenic epitope, we chemically coupled alpha-Gal (Galactose-a1,3-Galactose-b1,4-N-Acetylglucosamine) to specific sites of Ad capsomers. We generated ΔE1 Ad5 vectors being alpha-Galylated at hexon HVR1, hexon HVR5, penton or fiber, and an unmodified control vector.We demonstrated that on account of the alpha-Gal epitope, anti-Gal IgG or IgM bound to selected tagged capsomers. Thus, we analyzed biodistribution and vector-induced immune responses in alpha-1,3-Galactosyltransferase knockout (a1,3-GT KO) mice, which harbor large amounts of anti-Gal Abs.First, to compare the effect of epitope tagging on vector biodistribution, we analyzed hepatic and splenic transgene expression after i.v. injection of alpha-Galylated vectors into anti-Gal-positive a1,3-GT KO mice. We observed that transgene expression from fiber-modified vectors was mildly decreased, while expression from hexon-modified vectors decreased drastically, in a way resembling neutralization. These data suggested that, in fact, our model is suitable to precisely analyze the role of capsomer-specific Ab binding on in vivo vector biodistribution and activity.Furthermore, we analyzed the effect of immune complexation on the induction of vector- and transgene product-directed immune responses after repetitive i.m. delivery of alpha-Galylated vectors into anti-Gal-positive a1,3-GT KO mice. Here, we found that the Ad immune complexes induced transgene product-directed immune responses, even upon repeated vector administration. In particular, the extents of hepatic and splenic transgene expression, and transgene-directed immune responses were inversely correlated. For hexon-modified vectors, in spite of weakest transgene expression, transgene-directed cellular immune responses were strongest.In summary, we showed that alpha-Galylation of Ad capsomers markedly influenced specific immune responses directed to the vector as well as the transgene product. Our concept shall be used as an in vivo model to characterize whether directing or diverting immune complexation to or from specific capsomer sites can improve future strategies for vaccination regimens using Ad vectors.

Development of a Targeted Gene Vector Platform Based on Simian Adenovirus Serotype 24

Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations--widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery--justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.

Engineering Clustered Ligand Binding Into Nonviral Vectors: αvβ3 Targeting as an Example

The development of techniques to efficiently deliver genes using nonviral approaches can broaden the application of gene delivery in medical applications without the safety concerns associated with viral vectors. Here, we designed a clustered integrin-binding platform to enhance the efficiency and targetability of nonviral gene transfer to HeLa cells with low and high densities of alpha(v)beta(3) integrin receptors. Arg-Gly-Asp (RGD) nanoclusters were formed using gold nanoparticles functionalized with RGD peptides and used to modify the surface of DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes with attached RGD nanoclusters resulted in either 5.4- or 35-fold increase in gene transfer efficiency over unmodified polyplexes for HeLa cells with low- or high-integrin surface density, respectively. The transfection efficiency obtained with the commercially available vector jetPEI-RGD was used for comparison as a vector without clustered binding. JetPEI-RGD exhibited a 1.2-fold enhancement compared to unmodified jetPEI in cells with high densities of alpha(v)beta(3) integrin receptors. The data presented here emphasize the importance of the RGD conformational arrangement on the surface of the polyplex to achieve efficient targeting and gene transfer, and provide an approach to introduce clustering to a wide variety of nanoparticles for gene delivery.

Enhancement of Adenovirus-Mediated Gene Delivery to Rheumatoid Arthritis Synoviocytes and Synovium by Fiber Modifications: Role of Arginine-Glycine-Aspartic Acid (RGD)- and Non-RGD-Binding Integrins

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) do not express the coxsackie-adenovirus (Ad) receptor and are poorly permissive to Ad serotype 5 (Ad5). Genetically modified, coxsackie-Ad receptor-independent Ad5 vectors were studied for gene delivery in human RA FLS and synovium explants and murine collagen-induced arthritis. Short-fiber Ad5 vectors with seven fiber shaft repeats Ad5GFP-R7-knob, Ad5GFP-R7-arginine-glycine-aspartic acid (RGD) (RGD-liganded), and Ad5GFPDeltaknob (knob-deleted) were compared with Ad5GFP-FiWT, a conventional wild-type (WT) Ad5 vector. Gene transfer by Ad5GFP-R7-knob and Ad5GFP-R7-RGD was 40- to 50-fold and 25-fold higher, respectively, than Ad5GFP-FiWT in FLS. Ad5GFPDeltaknob was more efficacious than its knob-bearing version Ad5GFP-R7-knob in FLS transduction. Virus attachment and entry required RGD- and LDV-binding integrins including alpha(v), alpha(v)beta3, a(v)beta5, and beta1. Ad5GFP-R7-knob infection of FLS was partially neutralized by synovial fluid (SF), but remained 30- to 40-fold higher than Ad5GFP-FiWT in the presence of SF. Ad5GFPDeltaknob was partially neutralized by SF at low virus input, but escaped viral neutralization by SF at higher virus input. Gene transfer to human synovium ex vivo explants and murine collagen-induced arthritis in vivo was also more efficient with short fiber-modified vectors (with and without the knob domain) than Ad5GFPFiWT. Gene transfer by short fiber-modified vectors was enhanced by inflammatory cytokines in vitro and in the presence of inflammation in murine synovium in vivo. Our data indicated that the highly efficient gene delivery RA was mediated by RGD- and non-RGD-binding integrins and enhanced by inflammation. Short fiber modifications with knob ablation may be a strategy to enhance gene delivery, reducing vector dose and vector-induced inflammation and toxicity.

525. Optimized Gene Transfer into Mesenchymal Stem Cells and Embryonic Stem Cells by Modified Adenovirus Vectors

Mesenchymal stem cells (MSCs) and embryonic stem (ES) cells are considered a source of cells for regenerative medicine and for cell and gene therapy. Efficient gene transfer into MSCs and ES cells is essential for basic research into cellular differentiation and developmental biology and for therapeutic applications in gene-modified regenerative medicine. In this study, we optimized the transduction of MSCs and ES cells by means of various types of adenovirus (Ad) vectors. We optimized fiber-modifications and promoter sequences. Following fiber-modified vectors were used: Ad vectors containing an RGD peptide in the HI loop of the fiber knob; Ad vectors containing a polylysine peptide in the C-terminal of the fiber knob; and Ad vectors containing a fiber protein derived from the Ad type 35. For optimization of the promoter sequences, EF-1|[alpha]|, CMV, CMV with intron A, and CA (|[beta]|-actin promoter/CMV enhancer) promoters were tested. An Escherichia coli |[beta]|-galactosidase (LacZ) gene was used as a reporter gene.

Gene transfer to cervical cancer with fiber-modified adenoviruses.

Successful adenoviral (Ad) vector-mediated strategies for cancer gene therapy mandate gene-delivery systems that are capable of achieving efficient gene delivery in vivo. In many cancer types, in vivo gene-transfer efficiency remains limited due to the low or highly variable expression of the primary Ad receptor, the coxsackie Ad receptor (CAR). In this study, we evaluated the expression of CAR on cervical cancer cells as well as CAR-independent targeting strategies to integrins (Ad5.RGD), heparan sulfate proteoglycans (Ad5.pK7) or both (Ad5.RGD.pK7). We used a panel of established cervical cancer cell lines and primary cervical cancer cells isolated from patients to quantify the expression of CAR mRNA and to evaluate the gene-transfer efficiency of fiber-modified Ads. Of the fiber-modified vectors, Ad5.pK7 and Ad5.RGD.pK7 displayed significantly enhanced gene-transfer efficiency in vitro. Gene-delivery efficiency in vivo was evaluated using an s.c. cervical cancer mouse model. Ad5.RGD.pK7 significantly improves tumor targeting in vivo, resulting in a significantly improved tumor/liver ratio in mice. Our results suggest that the double-modified Ad5.RGD.pk7 vector enhances gene transfer to clinically relevant cervical cancer substrates, while the infectivity of nontarget cells in the mouse is not increased and comparable to Ad5. The fiber-modified virus described here can help achieve higher clinical efficacy of cervical cancer gene therapy.

Analysis of adenovirus sequestration in the liver, transduction of hepatic cells, and innate toxicity after injection of fiber-modified vectors.

After intravenous administration, adenovirus (Ad) vectors are predominantly sequestered by the liver. Delineating the mechanisms for Ad accumulation in the liver is crucial for a better understanding of Ad clearance and Ad-associated innate toxicity. To help address these issues, in this study, we used Ad vectors with different fiber shaft lengths and either coxsackievirus-Ad receptor (CAR)-interacting Ad serotype 9 (Ad9) or non-CAR-interacting Ad35 fiber knob domains. We analyzed the kinetics of Ad vector accumulation in the liver, uptake into hepatocytes and Kupffer cells, and induction of cytokine expression and release in response to systemic vector application. Immediately after intravenous injection, all Ad vectors accumulated equally efficiently in the liver; however, only genomes of long-shafted Ads were maintained in the liver tissue over time. We found that Kupffer cell uptake of long-shafted Ads was mediated by the fiber knob domain and was CAR independent. The short-shafted Ads were unable to efficiently interact with hepatocellular receptors and were not taken up by Kupffer cells. Moreover, our studies indicated that Kupffer cells were not the major reservoir for the observed accumulation of Ads (used in this study) in the liver within the first 30 min after virus infusion. The lower level of liver cell transduction by short-shafted Ads correlated with a significantly reduced inflammatory anti-Ad response as well as liver damage induced by the systemic administration of these vectors. This study contributes to a better understanding of the biology of systemically applied Ad and will help in designing safer vectors that can efficiently transduce target tissues.

Enhanced antitumor effect and reduced vector dissemination with fiber-modified adenovirus vectors expressing herpes simplex virus thymidine kinase.

There are at least two hurdles confronting the use of the adenovirus (Ad)-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system for the treatment of cancer. One is inefficient Ad vector-mediated gene transfer into tumor cells lacking the primary receptor, i.e., the coxsackievirus and adenovirus receptor (CAR). The other is hepatotoxicity due to unwanted vector spread into the liver, even when Ad vectors are injected intratumorally. Herein, we present an attractive strategy for overcoming such limitations based on use of a fiber-modified Ad vector containing an RGD peptide motif in the fiber knob. HSVtk-expressing Ad vectors containing mutant fiber (AdRGD-tk) or wild-type fiber (Ad-tk) were injected intratumorally into CAR-negative B16 melanoma cells inoculated into mice, after which GCV was injected intraperitoneally for 10 days. AdRGD-tk showed approximately 25 times more antitumor activity than Ad-tk. Histopathological studies suggested that liver damage in mice injected with AdRGD-tk was significantly lower than that in mice injected with Ad-tk. Intratumoral administration of luciferase-expressing Ad vectors containing the mutant fiber (AdRGD-L2) resulted in nearly 40 times more luciferase production in the tumor, but 8 times less production in the liver than the conventional Ad vectors (Ad-L2). These results indicate that combination of fiber-modified vectors and a HSVtk/GCV system is a potentially useful and safe approach for the treatment of tumors lacking CAR expression, and that fiber-modified vectors could be of great utility for gene therapy and gene transfer experiments.

A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob.

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.