Simon S. Briggs

A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids.

Synthetic vectors based on reducible polycations consisting of histidine and polylysine residues (HIS RPCs) were evaluated for their ability to deliver nucleic acids. Initial experiments showed that RPC-based vectors with at least 70% histidine content mediated efficient levels of gene transfer without requirement for the endosomolytic agent chloroquine. Significant gene transfer was observed in a range of cell types achieving up to a 5-fold increase in the percentage of transfected cells compared to 25 kDa PEI, a gold standard synthetic vector. In contrast to 25 kDa PEI, HIS RPCs also mediated efficient transfer of other nucleic acids, including mRNA encoding green fluorescent protein in PC-3 cells and siRNA directed against the neurotrophin receptor p75NTR in post-mitotic cultures of rat dorsal root ganglion cell neurons. Experiments to elevate intracellular glutathione and linear profiling of cell images captured by multiphoton fluorescent microscopy highlighted that parameters such as the molecular weight and rate of cleavage of HIS RPCs were important factors in determining transfection activity. Altogether, these results demonstrate that HIS RPCs represent a novel and versatile type of vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids. This should enable different or a combination of therapeutic strategies to be evaluated using a single type of polycation-based vector.

Polymer‐coated polyethylenimine/DNA complexes designed for triggered activation by intracellular reduction

Site‐specific gene delivery requires vectors that combine stability in the delivery phase with substantial biological activity within target cells. The use of biological trigger mechanisms provides one promising means to achieve this, and here we report a transfection trigger mechanism based on intracellular reduction.

Nonviral gene delivery: techniques and implications for molecular medicine.

Medical research continues to illuminate the origins of many human diseases. Gene therapy has been widely proposed as a novel strategy by which this knowledge can be used to deliver new and improved therapies. Viral gene transfer is relatively efficient but there are concerns relating to the use of viral vectors in humans. Conversely, nonviral vectors appear safe but inefficient. Therefore, the development of an efficient nonviral vector remains a highly desirable goal. This review focuses on the numerous challenges preventing efficient nonviral gene transfer in vivo and discusses the many technologies that have been adopted to overcome these problems.

Delivery of siRNA mediated by histidine-containing reducible polycations

Histidine containing reducible polycations based on CH(6)K(3)H(6)C monomers (His6 RPCs), are highly effective DNA transfection agents combining pH buffering endosomal escape mechanisms with rapid unpackaging following reduction in the cytoplasm. We examined their ability to mediate siRNA uptake into cells focusing on hepatocyte delivery. Co-delivery of EGFP siRNA with pEGFP plasmid DNA reduced reporter gene expression by 85%. However while DNA transfection efficiency increased with polymer size, with 162 k His6 RPCs proving the most effective, delivery of siRNA alone to EGFP stably expressing cells was only possible using 36-80 k polymers. Analysis of particle sizes showed that 80 k polymers formed more compact siRNA complexes than 162 k polymers. The reducible nature of the polymer was necessary for siRNA activity, since siRNA combined with non-reducible polylysine showed little activity. Incorporation of a targeting peptide from the Plasmodium falciparum circumsporozoite (CS) protein onto His6 RPCs, significantly improved transfection of plasmid DNA and siRNA activity in hepatocytes, but not in most non-liver cells tested. siRNA targeted to the hepatitis B virus surface antigen delivered by CS-His6 RPC, mediated falls in both mRNA and protein expression, suggesting that this delivery system could be developed for potential therapies for viral hepatitis.

Retargeting polymer-coated adenovirus to the FGF receptor allows productive infection and mediates efficacy in a peritoneal model of human ovarian cancer.

Transductional targeting of adenovirus following systemic or regional delivery remains one of the most difficult challenges for cancer gene medicine. The numerical excess and anatomical advantage of normal (non‐cancer) cells in vivo demand far greater detargeting than is necessary for studies using single cell populations in vitro, and this must be coupled with efficient retargeting to cancer cells.

Coating of adeno-associated virus with reactive polymers can ablate virus tropism, enable retargeting and provide resistance to neutralising antisera.

Copolymers based on poly‐[N‐(2‐hydroxypropyl) methacrylamide] (HPMA) have been used previously to enable targeted delivery of adenovirus. Here we demonstrate polymer‐coating techniques can also be used to modify and retarget adeno‐associated virus (AAV) types 5 and 8.

Cetuximab Retargeting of Adenovirus via the Epidermal Growth Factor Receptor for Treatment of Intraperitoneal Ovarian Cancer

Gene and virotherapy of ovarian cancer, using type 5 adenovirus (Ad5), has demonstrated good activity in preclinical animal studies, particularly after intraperitoneal administration of virus; however, success in clinical trials has been limited by poor infectivity of ovarian cancer cells and inflammatory responses to Ad5. We previously demonstrated that covalent modification of Ad5 with reactive copolymers on the basis of poly(hydroxypropylmethacrylamide) can shield the virus, offering protection from neutralizing antibodies and enabling retargeting to cancer-upregulated receptors with peptide ligands (basic fibroblast growth factor [bFGF] and murine epidermal growth factor [EGF]). These ligands may be less than ideal for clinical use, however, because they are potential mitogens. Accordingly, in this study we investigated the use of an anti-EGF receptor (EGFR) antibody, cetuximab, to retarget adenoviral transduction of EGFR-positives in vitro and in vivo. Cetuximab retargeting altered the physicochemical characteristics of Ad5, although it did not cause particle aggregation. Although cetuximab stimulated internalization of EGFR, similarly to EGF, it inhibited EGFR phosphorylation. Adenoviral transduction was inhibited after polymer coating, but was rescued in EGFR-positive cells (and not in EGFR-negative cells) by cetuximab retargeting. Cetuximab retargeting of wild-type adenovirus serotype 5 (Ad5WT) prolonged survival in an animal model of human ovarian cancer, similar to unmodified Ad5WT, but polymer coating ameliorated stimulation of adhesion formation. We conclude that polymer coating and covalent attachment of cetuximab successfully retargeted adenovirus to EGFR-positive cells, retained in vivo efficacy of an oncolytic adenovirus, and ameliorated side effects caused by unmodified adenovirus.

Multicomponent synthetic polymers with viral-mimetic chemistry for nucleic acid delivery.

The ability to deliver genetic material for therapy remains an unsolved challenge in medicine. Natural gene carriers, such as viruses, have evolved sophisticated mechanisms and modular biopolymer architectures to overcome these hurdles. Here we describe synthetic multicomponent materials for gene delivery, designed with features that mimic virus modular components and which transfect specific cell lines with high efficacy. The hierarchical nature of the synthetic carriers allows the incorporation of membrane-disrupting peptides, nucleic acid binding components, a protective coat layer, and an outer targeting ligand all in a single nanoparticle, but with functionality such that each is utilized in a specific sequence during the gene delivery process. The experimentally facile assembly suggests these materials could form a generic class of carrier systems that could be customized for many different therapeutic settings.

Use of synthetic vectors for neutralising antibody resistant delivery of replicating adenovirus DNA

Use of synthetic vectors to deliver genomes of conditionally replicating lytic viruses combines the strengths of viral and non-viral approaches by enabling neutralising antibody resistant deployment of cancer virotherapy. Adenovirus is particularly suitable for this application since all proteins essential for replication can be expressed from the input DNA, although the presence of terminal protein (TP) covalently linked to the 5′ termini of the input virus genomes both improves expression of transgenes encoded in the input DNA and also enhances replication. These roles of TP were distinguished in experiments where E1-deleted AdGFPDNA bearing TP (AdGFPDNA-TP), delivered with DOTAP, gave a two-fold greater frequency of transduction than AdGFPDNA(without TP) in non-complementing A549 cells, while in 293 cells (which support replication of E1-deleted viruses) the presence of TP mediated a much greater differential transgene expression, commensurate with its ability to promote replication. Subsequent studies using AdDNA for virotherapy, therefore, included covalently linked TP. AdDNA-TP delivered to A549 cells using a synthetic polyplex vector was shown to be resistant to levels of neutralising antisera that completely ablated infection by wild-type adenovirus, enabling polyplex/Adwild typeDNA-TP to mediate a powerful cytopathic effect. Similarly in vivo, direct injection of a polyplex/Adwild typeDNA-TP into A549 tumours was neutralising antibody-resistant and enabled virus replication, whereas intact virus was neutralised by the antibody and failed to infect. The delivery of adenovirus genomes–TP using synthetic vectors should provide a strategy to bypass neutralising antibodies and facilitate clinical application of replicating adenovirus for cancer virotherapy.

P‐selectin dependent targeting to inflamed endothelium of recombinant P‐selectin glycoprotein ligand‐1 immunoglobulin chimera‐coated poly[N‐(2‐hydroxypropyl) methacrylamide]‐DNA polyplexes in vivo visualised by intravital microscopy

Developing vectors that target specifically to disease sites after systemic injection is an important goal in gene therapy research.