Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control (original) (raw)
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Engineering polypeptide coatings to augment gene transduction and in vivo stability of adenoviruses
Journal of Controlled Release, 2013
We sought to modify adenoviral (Ad) particles by incorporating the advantageous characteristics of non-viral gene delivery vehicles to complement the viral vectors. α-Amino acid-N-carboxyanhydride chemistry was used to synthesize homopolypeptides and diblock copolypeptides that possess well-defined secondary structures. Using cryo-electron and fluorescence microscopy, we showed that these polypeptides can coat the surfaces of Ad particles in a non-covalent manner to modify their transduction properties. The coated Ad particles were found to bind to and be internalized by cells. In contrast to reports using covalently PEGylated Ad particles, we found that our physically coated Ad hybrid complexes facilitate gene transfer both in vitro and in vivo. We showed that our polypeptide coating was able to shield the Ad particles from the neutralizing effect of antibodies and mitigate the binding of blood coagulation factor (Factor X) in vitro. The coating also reduced the antigenicity of Ad in immunocompetent mice. The biodistribution of the systemically administered hybrid complexes mirrored the behavior of both viral and non-viral vectors, exhibiting liver tropism as well as enhanced lung transduction. These data demonstrated that our non-covalent modification was able to alter Ad's interactions with cells and organs with retention of transduction efficiency. Advantages such as facile coating of the Ad vector, design flexibility and ease of attaching ligands to the polypeptides make this system potentially useful as a platform for adding functionalities to Ad to target cancer metastasis.
Cancer Gene Therapy, 2007
In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.
Artificial envelopment of nonenveloped viruses: enhancing adenovirus tumor targeting in vivo
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2008
Recombinant adenovirus (Ad) is a powerful tool in gene therapy. However, the ability to deliver Ad by systemic administration is limited due to rapid clearance from blood circulation, transfection of nontarget tissues, toxicity, and immunogenicity. To address these limitations, we developed an artificially enveloped Ad vector prepared by self-assembly of lipid bilayers around the Ad capsid. The physicochemical and structural features of the enveloped Ad vector can be altered according to the type of lipid used without the need for genetic modification or conjugation chemistry. Here we engineered 4 different types of artificially enveloped Ad using cationic, neutral, fusogenic, and PEGylated lipids to form the envelopes and obtained extended blood circulation times following i.v. administration and reduced vector immunogenicity.
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) 1.35 nM) and fused this protein with a DARPin specific for Her2, an established cell-surface biomarker of human cancers. The stability of the complex formed by this bispecific targeting adapter and the Ad virion resulted in insufficient gene transfer and was subsequently improved by increasing the valency of adapter-virus binding. In particular, we designed adapters that chelated the knob in a bivalent or trivalent fashion and showed that the efficacy of gene transfer by the adapter-Ad complex increased with the functional affinity of these molecules. This enabled efficient transduction at low stoichiometric adapter-to-fiber ratios. We confirmed the Her2 specificity of this transduction and its dependence on the Her2-binding DARPin component of the adapters. Even the adapter molecules with four fused DARPins could be produced and purified from Escherichia coli at very high levels. In principle, DARPins can be generated against any target and this adapter approach provides a versatile strategy for developing a broad range of disease-specific gene vectors.
Journal of Virology, 2008
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. This shortage is due to fundamental and irresolvable differences at the level of both posttranslational modifications and intracellular trafficking between the Ad structural proteins and those natural proteins that are involved in interactions with the cell surface and could otherwise be considered as potential targeting ligands. We hypothesized that this problem could be resolved by altering the natural tropism of Ad vector through incorporation into its capsid of a rationally designed protein ligand, an affibody, whose structural, functional, and biosynthetic properties make it compatible with the Ad assembly process. We tested this hypothesis by redesigning the receptor-binding Ad protein, the fiber, using affibodies specific for human epidermal growth factor receptor type 2 (Her2), a major molecular marker of human tumors. The biosynthesis and folding of these fiber chimeras were fully compatible with Ad virion formation, and the resultant viral vectors were capable of selective delivery of a dual-function transgene to Her2expressing cancer cells. By establishing the feasibility of this affibody-based approach to Ad vector targeting, the present study lays the foundation for further development of Ad vector technology toward its clinical use.
Journal of the American Chemical Society, 2010
Azide-enabled adenovirus for gene delivery: We demonstrate here a novel two-step "click" labeling process in which adenoviral particles are first metabolically labeled during production with unnatural azido sugars. Subsequent chemoselective modification allows access to viruses decorated with a broad array of effector functionality. Adenoviruses modified with folate, a known cancer-targeting motif, demonstrated a marked increase in gene delivery to a murine cancer cell line. Virus mediated gene delivery has been limited by the inability to easily access a wide array of surface displayed functionality. Here we demonstrate the chemoselective attachment of a cancer selective small molecule, folate, to the surface of human adenovirus type 5 (hAd5). Modification of the viral surface is mediated by the metabolic incorporation of an azido sugar, O-linked N-acetylglucosamine (O-GlcNAz), into the fiber protein during virus production. Folate decorated hAd5 demonstrate a significant increase in transgene delivery to murine breast cancer cells. Viral surface engineering has significant potential to tune the virus-host-interface enabling gene replacement therapy, oncolytic therapy and vaccine development. 1-3 Despite being a long-standing objective, targeting therapeutically relevant tissues remains a significant hurdle. 2 A contributing factor is the lack of general, effective methods to introduce targeting ligands onto viral surfaces that do not compromise virus production or infectivity. Currently the majority of targeting efforts rely upon genetic manipulation of virus surface proteins. However, due to the delicate nature of viral protein assembly and infection, such changes
The ‘adenobody’ approach to viral targeting: specific and enhanced adenoviral gene delivery
Gene Therapy, 1997
Recombinant adenoviruses have enormous potential as system the efficiency of viral infection was markedly vectors for gene therapy. They have evolved an efficient enhanced and was targeted to the EGFR. The adenobodymethod of infection and a wide host range but this leads directed infection correlated with the level of EGF receptor to concerns about the specificity of gene delivery. In order expressed on the cells and could be blocked by competo target an adenovirus type 5-based vector we have inves-tition with pure EGF. Peptide inhibition experiments sugtigated an antibody approach. A virus neutralising scFv gest that infection is mediated directly through attachment antibody fragment was isolated from a phage library and a to the EGFR and does not require penton-integrin inter-C-terminal fusion protein with epidermal growth factor actions. This work shows that the 'adenobody' approach (EGF) constructed. This fusion protein, or 'adenobody', can enhance the efficiency as well as target adenoviral bound both to the fibre protein of the adenovirus and to the infection and has numerous potential applications for EGF receptor (EGFR) on human cells, and was able to gene therapy. direct adenoviral binding to the new receptor. Using this process with virion attachment a distinct and dissociable Correspondence: RE Hawkins, Bristol University Department of that the scFv-EGF fusion protein could both target and Oncology, Bristol Oncology Centre, Horfield Road, Bristol, BS2 8ED, UK enhance the efficiency of adenoviral gene delivery. The 3 Current address: Shemyakin-Ovchnnikov Institute of Bio-organic Chemmechanisms involved were explored. istry,
Adenovirus – a blueprint for gene delivery
Current Opinion in Virology, 2021
A central quest in gene therapy and vaccination is to achieve effective and long-lasting gene expression at minimal dosage. Adenovirus vectors are widely used therapeutics and safely deliver genes into many cell types. Adenoviruses evolved to use elaborate trafficking and particle deconstruction processes, and efficient gene expression and progeny formation. Here, we discuss recent insights into how human adenoviruses deliver their double-stranded DNA genome into cell nuclei, and effect lytic cell killing, non-lytic persistent infection or vector gene expression. The mechanisms underlying adenovirus entry, uncoating, nuclear transport and gene expression provide a blueprint for the emerging field of synthetic virology, where artificial virus-like particles are evolved to deliver therapeutic payload into human cells without viral proteins and genomes.
Silica cloaking of adenovirus enhances gene delivery while reducing immunogenicity
Journal of Controlled Release, 2019
Viral gene therapy is a means of delivering genes to replace malfunctioning ones, to kill cancer cells, or to correct genetic mutations. This technology is emerging as a powerful clinical tool; however, it is still limited by viral tropism, uptake and clearance by the liver, and most importantly an immune response. To overcome these challenges, we sought to merge the
Journal of Virology
Adeno-associated virus serotype 5 (AAV5) is being developed as a gene delivery vector for several diseases, including hemophilia and Huntington’s disease, and has a demonstrated efficient transduction in liver, lung, skeletal muscle, and the central nervous system. One limitation of AAV gene delivery is preexisting neutralizing antibodies, which present a significant challenge for vector effectiveness in therapeutic applications. Here, we report the cryo-electron microscopy (cryo-EM) and image-reconstructed structure of AAV5 in complex with a newly generated monoclonal antibody, HL2476, at 3.1-Å resolution. Unlike other available anti-AAV5 capsid antibodies, ADK5a and ADK5b, with epitopes surrounding the 5-fold channel of the capsid, HL2476 binds to the 3-fold protrusions. To elucidate the capsid-antibody interactions, the heavy and light chains were sequenced and their coordinates, along with the AAV5 viral protein, assigned to the density map. The high resolution of the complex en...