Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43.

Unique molecular properties of species D adenoviruses (Ads)-the most diverse yet underexplored group of Ads-have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization.

Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters.

Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice.

Molecular imaging of active mutant L858R EGF receptor (EGFR) kinase-expressing nonsmall cell lung carcinomas using PET/CT.

The importance of the EGF receptor (EGFR) signaling pathway in the development and progression of nonsmall cell lung carcinomas (NSCLC) is widely recognized. Gene sequencing studies revealed that a majority of tumors responding to EGFR kinase inhibitors harbor activating mutations in the EGFR kinase domain. This underscores the need for novel biomarkers and diagnostic imaging approaches to identify patients who may benefit from particular therapeutic agents and approaches with improved efficacy and safety profiles.

Her2-specific multivalent adapters confer designed tropism to adenovirus for gene targeting.

Adenoviruses (Ads) hold great promise as gene vectors for diagnostic or therapeutic applications. The native tropism of Ads must be modified to achieve disease site-specific gene delivery by Ad vectors and this should be done in a programmable way and with technology that can realistically be scaled up. To this end, we applied the technologies of designed ankyrin repeat proteins (DARPins) and ribosome display to develop a DARPin that binds the knob domain of the Ad fiber protein with low nanomolar affinity (K(D) 1.

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).

A strategy for adenovirus vector targeting with a secreted single chain antibody.

Background: Successful gene therapy will require targeted delivery vectors capable of self-directed localization. In this regard, the use of antibodies or single chain antibody fragments (scFv) in conjunction with adenovirus (Ad) vectors remains an attractive means to achieve cell-specific targeting. However, a longstanding barrier to the development of Ad vectors with genetically incorporated scFvs has been the biosynthetic incompatibility between Ad capsid proteins and antibody-derived species.

Therapeutic Molecular Targetingof 15-Lipoxygenase-1 in Colon Cancer.

Molecular targeting for apoptosis induction is being developed for better treatment of cancer. Downregulation of 15-lipoxygenase-1 (15-LOX-1) is linked to colorectal tumorigenesis. Re-expression of 15-LOX-1 in cancer cells by pharmaceutical agents induces apoptosis.

Therapeutic molecular targeting of 15-lipoxygenase-1 in colon cancer.

Molecular targeting for apoptosis induction is being developed for better treatment of cancer. Downregulation of 15-lipoxygenase-1 (15-LOX-1) is linked to colorectal tumorigenesis. Re-expression of 15-LOX-1 in cancer cells by pharmaceutical agents induces apoptosis.

Modification of adenovirus capsid with a designed protein ligand yields a gene vector targeted to a major molecular marker of cancer.

The future of genetic interventions in humans critically depends on the selectivity and efficiency of gene transfer to target tissues. The viral gene vectors explored to date cannot selectively transduce the desired targets. While substantial progress has been made in developing targeting strategies for adenovirus (Ad) vectors, future advances in this direction are severely limited by the shortage of naturally existing molecules available for use as targeting ligands.

Cell cycle arrest and apoptosis induced by SART-1 gene transduction.

Unlabelled: The biological function of the SART-1 gene product is demonstrated and its potential as a target for cancer gene therapy is discussed.

Materials And Methods: The SART-1 gene was transduced by a recombinant adenovirus vector and its expression was promoted by a CMV promoter.

Results: The transduction efficiency by recombinant adenoviruses in A549 and MCF-7 cells was determined using a vector expressing luciferase, which showed high expression in the cells.

Identification of sites in adenovirus hexon for foreign peptide incorporation.

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive.

Promoter-controlled infectivity-enhanced conditionally replicative adenoviral vectors for the treatment of gastric cancer.

Background: Gastric cancer is the fourth most common malignancy worldwide. Adenoviral vectors (Ads) have been applied for gene therapy of various cancers because of their high transduction efficiency. However, the infectivity of gastrointestinal cancer cells is poor due to the limited expression of the Coxsackie-adenovirus receptor (CAR).

Adenoviruses with an RGD-4C modification of the fiber knob elicit a neutralizing antibody response but continue to allow enhanced gene delivery.

Objective: The purpose of this study was to investigate the effect of preexisting neutralizing antibody (NAbs) in naive mice and the effect of induced NAbs in mice immunized with either an RGD or nonmodified Ad5 vector on the transduction efficiency of adenoviral vectors.

Methods: BALB/c mice were immunized with Ad5LucRGD, with the unmodified Ad5Luc1, or with Opti-MEM intraperitoneally (ip) from one to three times. Sera were collected on day 27 and serially diluted to block Ad5Luc1 or Ad5LucRGD prior to infection of SKOV3.

Infectivity-enhanced cyclooxygenase-2-based conditionally replicative adenoviruses for esophageal adenocarcinoma treatment.

The employment of conditionally replicative adenoviruses (CRAd) constitutes a promising alternative for cancer treatment; however, in the case of esophageal adenocarcinoma (EAC) the lack of an appropriate tumor-specific promoter and relative resistance to adenovirus infection have hampered the construction of CRAds with clinically applicable specificity and efficacy. By combining transcriptional targeting with infectivity enhancement for CRAds, we generated novel cyclooxygenase-2 (Cox-2) promoter-controlled replicative viral agents for the treatment of EAC. We used infectivity enhancement based on incorporation of an RGD-4C motif into the HI loop of the adenoviral (Ad) fiber knob domain as well as replacement of the Ad5 knob with the Ad3 knob.

HSV-1 virions engineered for specific binding to cell surface receptors.

Expression of specific peptide epitopes on the surface of virions has significant potential for studying viral biology and designing vectors for targeted gene therapy. In this study, an HSV-1 amplicon plasmid expressing a modified glycoprotein C (gC), in which the heparan sulfate binding domain was replaced with a His-tag, was used in generating HSV-1 virions. Western blot analysis demonstrated the presence of modified gC in the purified virions.

Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo.

The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdDeltaE1AIL), the early gene E2B (AdDeltaE2BIL), or the late fiber gene (AdDeltafiberIL).

Adenovirus-mediated gene delivery to dendritic cells.

Dendritic cells (DCs) are "professional" antigen-presenting cells (APCs) that are uniquely capable of activating and instructing a naive immune system to mount a specific cellular and humoral response. Recognition of this crucial function makes the development of technologies for DC-based immuno-therapies a priority for the treatment of a wide variety of diseases. The most immediate impact of this emerging technology will be in the treatment of cancer and the development of third generation vaccines to protect against viral and intracellular pathogens.

Targeting of adenovirus via genetic modification of the viral capsid combined with a protein bridge.

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.

Genetically targeted adenovirus vector directed to CD40-expressing cells.

The success of gene therapy depends on the specificity of transgene delivery by therapeutic vectors. The present study describes the use of an adenovirus (Ad) fiber replacement strategy for genetic targeting of the virus to human CD40, which is expressed by a variety of diseased tissues. The tropism of the virus was modified by the incorporation into its capsid of a protein chimera comprising structural domains of three different proteins: the Ad serotype 5 fiber, phage T4 fibritin, and the human CD40 ligand (CD40L).

Infectivity enhanced, cyclooxygenase-2 promoter-based conditionally replicative adenovirus for pancreatic cancer.

Background And Aims: Pancreatic cancer is one of the most aggressive human malignancies. Conditionally replicative adenoviruses (CRAds) have shown some promise in the treatment of cancers. However, to date, their application for pancreatic cancer has met several obstacles: one is lack of a good control element to regulate replication, and the other is relatively low adenoviral infectivity.

A mosaic adenovirus possessing serotype Ad5 and serotype Ad3 knobs exhibits expanded tropism.

The efficiency of cancer gene therapy with recombinant adenoviruses based on serotype 5 (Ad5) has been limited partly because of variable, and often low, expression by human primary cancer cells of the primary cellular-receptor which recognizes the knob domain of the fiber protein, the coxsackie and adenovirus receptor (CAR). As a means of circumventing CAR deficiency, Ad vectors have been retargeted by utilizing chimeric fibers possessing knob domains of alternate Ad serotypes. We have reported that ovarian cancer cells possess a primary receptor for Ad3 to which the Ad3 knob binds independently of the CAR-Ad5 knob interaction.

Blood-based screening and light based imaging for the early detection and monitoring of ovarian cancer xenografts.

We report a novel technology for in vivo early detection, identification, and monitoring of ovarian cancer in live mice leading to better treatment outcome. A genetic dualistic reporter system that uses an adenoviral (Ad) vector to transfer the genetic reporters to the ovarian cancer is described. Infection of the cancer cells leads to expression of one reporter that is detected in blood, namely, secreted human placental alkaline phosphatase (SEAP).

Development of a therapeutic adenoviral vector for cholangiocarcinoma combining tumor-restricted gene expression and infectivity enhancement.

Cholangiocarcinoma is an invasive malignancy that is most often unresectable upon diagnosis and unresponsive to chemotherapy and radiation. While adenoviral gene therapy has shown promise in treating many tumors, systemic toxicity and low tumor transduction efficiency have hampered its application in many gastrointestinal cancers. To overcome these difficulties, we have constructed an adenoviral vector utilizing a tumor-specific promoter (TSP) for selective transgene expression and a vector with an RGD-motif in the fiber-knob region for infectivity enhancement.

Substitution of the adenovirus serotype 5 knob with a serotype 3 knob enhances multiple steps in virus replication.

Adenovirus (Ad) serotype 5 (Ad5) continues to be the predominant vector used for cancer gene therapy. However, many tumor types are reported to be relatively refractory to Ad5 infection because of low surface expression of the native Ad5 receptor, CAR. The observation that many tumor cells are CAR deficient has necessitated the development of CAR-independent infection strategies, including the introduction of heterologous ligand sequences into the virus fiber gene and immunological or chemical modifications of the capsid proteins.

Modulation of adenovirus vector tropism via incorporation of polypeptide ligands into the fiber protein.

The efficacy of adenovirus (Ad)-based gene therapy might be significantly improved if viral vectors capable of tissue-specific gene delivery could be developed. Previous attempts to genetically modify the tropism of Ad vectors have been only partially successful, largely due to the limited repertoire of ligands that can be incorporated into the Ad capsid. Early studies identified stringent size limitations imposed by the structure of the Ad fiber protein on ligands incorporated into its carboxy terminus and thus limited the range of potential ligand candidates to short peptides.