Nigel A.J. McMillan

Lipidic Systems for In Vivo siRNA Delivery

The ability of small-interfering RNA (siRNA) to silence specific target genes not only offers a tool to study gene function but also represents a novel approach for the treatment of various human diseases. Its clinical use, however, has been severely hampered by the lack of delivery of these molecules to target cell populations in vivo due to their instability, inefficient cell entry, and poor pharmacokinetic profile. Various delivery vectors including liposomes, polymers, and nanoparticles have thus been developed in order to circumvent these problems. This review presents a comprehensive overview of the barriers and recent progress for both local and systemic delivery of therapeutic siRNA using lipidic vectors. Different strategies for formulating these siRNA-loaded lipid particles as well as the general concern about their safe use in vivo will also be discussed. Finally, current advances in the targeted delivery of siRNA and their impacts on the field of RNA interference (RNAi)-based therapy will be presented.

Potential strategies utilised by papillomavirus to evade host immunity.

Summary: The co‐evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non‐specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus‐induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced lo PV antigens, resting keratinocytes (KC) appear resistant to interferon‐γ‐enhanced mechanisms of cytotoxic T‐lymphocyte (CTL)‐mediated lysis, and expression of PV antigens by resting KC can tolerise PV‐specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV‐induced proliferative skin lesions for months to years, even in immunocompetent hosts.

Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into Rag−/− mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.

MicroRNAs regulate tumor angiogenesis modulated by endothelial progenitor cells

Bone marrow-derived endothelial progenitor cells (EPC) contribute to the angiogenesis-dependent growth of tumors in mice and humans. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. miRNAs have emerged as key regulators of several cellular processes including angiogenesis; however, whether miRNAs contribute to bone marrow-mediated angiogenesis has remained unknown. Here, we show that genetic ablation of miRNA-processing enzyme Dicer, specifically in the bone marrow, decreased the number of circulating EPCs, resulting in angiogenesis suppression and impaired tumor growth. Furthermore, genome-wide deep sequencing of small RNAs revealed tumor EPC-intrinsic miRNAs including miR-10b and miR-196b, which have been previously identified as key regulators of HOX signaling and adult stem cell differentiation. Notably, we found that both miR-10b and miR-196b are responsive to vascular endothelial growth factor stimulation and show elevated expression in human high-grade breast tumor vasculature. Strikingly, targeting miR-10b and miR-196b led to significant defects in angiogenesis-mediated tumor growth in mice. Targeting these miRNAs may constitute a novel strategy for inhibiting tumor angiogenesis.

Rational Design of Immunostimulatory siRNAs

Short-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on their sequence, can be variably sensed by the innate immune system through recruitment of toll-like receptors 7 and 8 (TLR7/8). Here, we aimed to identify sequence-based modifications allowing for the design of bifunctional siRNAs with both proinflammatory and specific silencing activities, and with potentially increased therapeutic benefits. We found that the introduction of a micro-RNA (miRNA)-like nonpairing uridine-bulge in the passenger strand robustly increased immunostimulatory activity on human immune cells. This sequence modification had no effect on the silencing efficiency of the siRNA. Increased immunostimulation with the uridine-bulge design was specific to human cells, and conserved silencing efficiency required a Dicer-substrate scaffold. The increased cytokine production with the uridine-bulge design resulted in enhanced protection against Semliki Forest virus (SFV) infection, in viral assays. Thus, we characterize a design scaffold applicable to any given siRNA sequence, that results in increased innate immune activation without affecting gene silencing. Our data suggest that this sequence modification coupled with structural modification differentially recruits human TLR8 over TLR7, and could have potential application in antiviral therapies.

Dendrimer nanocarriers as versatile vectors in gene delivery

UNLABELLED The successful delivery of nucleic acids to particular target sites is the challenge that is being addressed using a variety of viral and nonviral delivery systems, both of which have distinct advantages and disadvantages. Nonviral vectors offer the advantage of safety and flexibility over viral vectors, although they lack efficiency. Dendrimers are novel, three-dimensional polymers that have the ability to interact with various forms of nucleic acids such as plasmid DNA, antisense oligonucleotides, and RNA to form complexes that protect the nucleic acid from degradation. The interaction between the dendrimers and the nucleic acids is purely electrostatic where the cationic dendrimer condenses the anionic nucleic acids. Because cell membranes are negatively charged, the net positive charge of the dendrimer nucleic acid complex determines the transfection efficiency, although highly cationic systems are also cytotoxic. The nature of the dendrimer nucleic acid complex depends on various factors like stoichiometry, concentration of dendrimer-amines and nucleic acid-phosphates, as well as bulk solvent properties like pH, salt concentration, buffer strength, and dynamics of mixing. This article aims to review the role of dendrimers as novel gene delivery vectors both in vitro and in vivo. Dendrimer-based transfection reagents have become routine tools for in vitro transfection, but in vivo delivery of therapeutic nucleic acids remains a challenge. FROM THE CLINICAL EDITOR This review discusses the role of dendrimers as novel gene delivery vectors both in vitro and in vivo. Dendrimer based transfection reagents have become routine tools for in vitro transfection but in vivo delivery of therapeutic nucleic acids remains a challenge.

Vaginal delivery of siRNA using a novel PEGylated lipoplex-entrapped alginate scaffold system

Sustained vaginal delivery of siRNA has been precluded by the mucosal barrier lining the vaginal tract. In contrast to prior reports, we showed that conventional lipoplexes administered intravaginally are unable to reach the vaginal epithelium under normal physiological conditions. Here we have developed a novel alginate scaffold system containing muco-inert PEGylated lipoplexes to provide a sustained vaginal presence of lipoplexes in vivo and to facilitate the delivery of siRNA/oligonucleotides into the vaginal epithelium. These PEGylated lipoplex-entrapped alginate scaffolds (PLAS) were fabricated using a freeze-drying method and the entrapment efficiency, release rate, and efficacy were characterized. We demonstrated that the PLAS system had an entrapment efficiency of ~50%, which released PEGylated lipoplexes gradually both in vitro and in vivo. While the presence of alginate diminished the cell uptake efficiency of PEGylated lipoplexes in vitro, as expected, we showed a six-fold increase their uptake into the vaginal epithelium compared to existing transfection systems following intravaginal administration in mice. A significant knockdown of Lamin A/C level was also observed in vaginal tissues using siLamin A/C-containing PLAS system in vivo. Overall, our results indicated the potential of the biodegradable PLAS system for the sustained delivery of siRNA/oligonucleotides to vaginal epithelium.

RETRACTED: Dendrimer nanocarriers as versatile vectors in gene delivery

UNLABELLED The successful delivery of nucleic acids to particular target sites is the challenge that is being addressed using a variety of viral and nonviral delivery systems, both of which have distinct advantages and disadvantages. Nonviral vectors offer the advantage of safety and flexibility over viral vectors, although they lack efficiency. Dendrimers are novel, three-dimensional polymers that have the ability to interact with various forms of nucleic acids such as plasmid DNA, antisense oligonucleotides, and RNA to form complexes that protect the nucleic acid from degradation. The interaction between the dendrimers and the nucleic acids is purely electrostatic where the cationic dendrimer condenses the anionic nucleic acids. Because cell membranes are negatively charged, the net positive charge of the dendrimer nucleic acid complex determines the transfection efficiency, although highly cationic systems are also cytotoxic. The nature of the dendrimer nucleic acid complex depends on various factors like stoichiometry, concentration of dendrimer-amines and nucleic acid-phosphates, as well as bulk solvent properties like pH, salt concentration, buffer strength, and dynamics of mixing. This article aims to review the role of dendrimers as novel gene delivery vectors both in vitro and in vivo. Dendrimer-based transfection reagents have become routine tools for in vitro transfection, but in vivo delivery of therapeutic nucleic acids remains a challenge. FROM THE CLINICAL EDITOR This review discusses the role of dendrimers as novel gene delivery vectors both in vitro and in vivo. Dendrimer based transfection reagents have become routine tools for in vitro transfection but in vivo delivery of therapeutic nucleic acids remains a challenge.

Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC(50) (50% inhibition) was only 0.5 microg/ml for heparin, 1 microg/ml for dextran sulfate, and 5-10 microg/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate.

Human papillomavirus type spectrum in normal skin of individuals with or without a history of frequent sun exposure

Cutaneous human papillomavirus (HPV) has been widely detected in healthy skin. Previous studies have found that UV radiation can activate several HPV types, and a possible role for cutaneous HPV in the development of non-melanoma skin cancer has been suggested. This study investigated the prevalence and type-spectrum of cutaneous HPV in relation to UV radiation by studying forehead skin swab samples from 50 healthy males frequently exposed to the sun and 50 healthy males who were not frequently exposed to the sun. A questionnaire including ethnic background of the participants, history of cancers and a self-assessment of sun-exposure was also conducted and analysed. PCR with the FAP primer pair was carried out to detect HPV DNA in samples. HPV prevalence was higher in individuals who spent more time outdoors and in individuals with a history of skin cancers (P=0.044 and P=0.04, respectively). Furthermore, individuals wearing sunglasses as a means of sun protection had a lower prevalence of HPV (P=0.018). Interestingly, HPV-76 was only detected in the group without frequent sun-exposure (P=0.001). These results suggest that increased UV radiation exposure may be a factor leading to a difference in prevalence of cutaneous HPV types.