Shear-thinning hydrogel for allograft cell transplantation and externally controlled transgene expression.

This work establishes the design of a fully synthetic, shear-thinning hydrogel platform that is injectable and can isolate engineered, allogeneic cell therapies from the host. We utilized RAFT to generate a library of linear random copolymers of N,N-dimethylacrylamide (DMA) and 2-vinyl-4,4-dimethyl azlactone (VDMA) with variable mol% VDMA and degree of polymerization. Poly(DMA-co-VDMA) copolymers were subsequently modified with either adamantane (Ad) or ß-cyclodextrin (Cd) through amine-reactive VDMA to prepare hydrogel precursor macromers containing complementary guest-host pairing pendant groups that, when mixed, form shear-thinning hydrogels. Rheometric evaluation of the hydrogel library enabled identification of lead macromer structures comprising 15 mol% pendants (Ad or Cd) and a degree of polymerization of 1000; mixing of these Ad and Cd functionalized precursors yielded hydrogels possessing storage modulus above 1000 Pa, tan(δ) values below 1 and high yield strain, which are target characteristics of robust but injectable shear-thinning gels. This modular system proved amenable to nanoparticle integration with surface-modified nanoparticles displaying Ad. The addition of the Ad-functionalized nanoparticles simultaneously improved mechanical properties of the hydrogels and enabled extended hydrogel retention of a model small molecule in vivo. In studies benchmarking against alginate, a material traditionally used for cell encapsulation, the lead hydrogel showed significantly less fibrous encapsulation in a subcutaneous implant site. Finally, this platform was utilized to encapsulate and extend in vivo longevity of inducible transgene-engineered mesenchymal stem cells in an allogeneic transplant model. The hydrogels remained intact and blocked infiltration by host cells, consequently extending the longevity of grafted cell function relative to a benchmark, shear-thinning hyaluronic acid-based gel. In sum, the new synthetic, shear-thinning hydrogel system presented here shows potential for further development as an injectable platform for delivery and in situ drug modulation of allograft and engineered cell therapies.

Body size modulates the extent of seasonal diet switching by large mammalian herbivores in Yellowstone National Park.

Prevailing theories about animal foraging behaviours and the food webs they occupy offer divergent predictions about whether seasonally limited food availability promotes dietary diversification or specialization. Emphasis on how animals compete for food predominates in work on the foraging ecology of large mammalian herbivores, whereas emphasis on how the diversity of available foods generally constrains dietary opportunity predominates work on entire food webs. Reconciling predictions about what promotes dietary diversification is challenging because species' different body sizes and mobilities modulate how they seek and compete for resources-the mechanistic bases of common predictions may not pertain to all species equally. We evaluated predictions about five large-herbivore species that differ in body size and mobility in Yellowstone National Park using GPS tracking and dietary DNA. The data illuminated remarkably strong and significant correlations between body size and five key indicators of diet seasonality (R 2 = 0.71-0.80). Compared to smaller species, bison and elk showed muted diet seasonality and maintained access to more unique foods when winter conditions constrained food availability. Evidence from GPS collars revealed size-based differences in species' seasonal movements and habitat-use patterns, suggesting that better accounting for the allometry of foraging behaviours may help reconcile disparate ideas about the ecological drivers of seasonal diet switching.

Structural optimization of siRNA conjugates for albumin binding achieves effective MCL1-directed cancer therapy.

The high potential of siRNAs to silence oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, divalent lipid-conjugated siRNAs are optimized for in situ binding to albumin to improve pharmacokinetics and tumor delivery. Systematic variation of the siRNA conjugate structure reveals that the location of the linker branching site dictates tendency toward albumin association versus self-assembly, while the lipid hydrophobicity and reversibility of albumin binding also contribute to siRNA intracellular delivery. The lead structure increases tumor siRNA accumulation 12-fold in orthotopic triple negative breast cancer (TNBC) tumors over the parent siRNA. This structure achieves approximately 80% silencing of the anti-apoptotic oncogene MCL1 and yields better survival outcomes in three TNBC models than an MCL-1 small molecule inhibitor. These studies provide new structure-function insights on siRNA-lipid conjugate structures that are intravenously injected, associate in situ with serum albumin, and improve pharmacokinetics and tumor treatment efficacy.

Engineering approaches for RNA-based and cell-based osteoarthritis therapies.

Osteoarthritis (OA) is a chronic, debilitating disease that substantially impairs the quality of life of affected individuals. The underlying mechanisms of OA are diverse and are becoming increasingly understood at the systemic, tissue, cellular and gene levels. However, the pharmacological therapies available remain limited, owing to drug delivery barriers, and consist mainly of broadly immunosuppressive regimens, such as corticosteroids, that provide only short-term palliative benefits and do not alter disease progression. Engineered RNA-based and cell-based therapies developed with synthetic chemistry and biology tools provide promise for future OA treatments with durable, efficacious mechanisms of action that can specifically target the underlying drivers of pathology. This Review highlights emerging classes of RNA-based technologies that hold potential for OA therapies, including small interfering RNA for gene silencing, microRNA and anti-microRNA for multi-gene regulation, mRNA for gene supplementation, and RNA-guided gene-editing platforms such as CRISPR-Cas9. Various cell-engineering strategies are also examined that potentiate disease-dependent, spatiotemporally regulated production of therapeutic molecules, and a conceptual framework is presented for their application as OA treatments. In summary, this Review highlights modern genetic medicines that have been clinically approved for other diseases, in addition to emerging genome and cellular engineering approaches, with the goal of emphasizing their potential as transformative OA treatments.

Albumin-binding RNAi Conjugate for Carrier Free Treatment of Arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are joint diseases that are associated with pain and lost quality of life. No disease modifying OA drugs are currently available. RA treatments are better established but are not always effective and can cause immune suppression. Here, an MMP13-selective siRNA conjugate was developed that, when delivered intravenously, docks onto endogenous albumin and promotes preferential accumulation in articular cartilage and synovia of OA and RA joints. MMP13 expression was diminished upon intravenous delivery of MMP13 siRNA conjugates, consequently decreasing multiple histological and molecular markers of disease severity, while also reducing clinical manifestations such as swelling (RA) and joint pressure sensitivity (RA and OA). Importantly, MMP13 silencing provided more comprehensive OA treatment efficacy than standard of care (steroids) or experimental MMP inhibitors. These data demonstrate the utility of albumin 'hitchhiking' for drug delivery to arthritic joints, and establish the therapeutic utility of systemically delivered anti-MMP13 siRNA conjugates in OA and RA. Editorial summary: Lipophilic siRNA conjugates optimized for albumin binding and "hitchhiking" can be leveraged to achieve preferential delivery to and gene silencing activity within arthritic joints. Chemical stabilization of the lipophilic siRNA enables intravenous siRNA delivery without lipid or polymer encapsulation. Using siRNA sequences targeting MMP13, a key driver of arthritis-related inflammation, albumin hitchhiking siRNA diminished MMP13, inflammation, and manifestations of osteoarthritis and rheumatoid arthritis at molecular, histological, and clinical levels, consistently outperforming clinical standards of care and small molecule MMP antagonists.

Core polymer optimization of ternary siRNA nanoparticles enhances in vivo safety, pharmacokinetics, and tumor gene silencing.

Gene silencing with siRNA nanoparticles (si-NPs) is promising but still clinically unrealized for inhibition of tumor driver genes. Ternary si-NPs containing siRNA, a single block NP core-forming polymer poly[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] (DMAEMA-co-BMA, 50B), and an NP surface-forming diblock polymer 20 kDa poly(ethylene glycol)-block-50B (20kPEG-50B) have the potential to improve silencing activity in tumors due to the participation of both 50B and 20kPEG-50B in siRNA electrostatic loading and endosome disruptive activity. Functionally, single block 50B provides more potent endosomolytic activity, while 20kPEG-50B colloidally stabilizes the si-NPs. Here, we systematically explored the role of the molecular weight (MW) of the core polymer and of the core:surface polymer ratio on ternary si-NP performance. A library of ternary si-NPs was formulated with variation in the MW of the 50B polymer and in the ratio of the core and surface forming polymeric components. Increasing 50B core polymer MW and ratio improved si-NP in vitro gene silencing potency, endosome disruptive activity, and stability, but these features also correlated with cytotoxicity. Concomitant optimization of 50B size and ratio resulted in the identification of lead ternary si-NPs 50B4-DP100, 50B8-DP100, and 50B12-DP25, with potent activity and minimal toxicity. Following intravenous treatment in vivo, all lead si-NPs displayed negligible toxicological effects and enhanced pharmacokinetics and tumor gene silencing relative to more canonical binary si-NPs. Critically, a single 1 mg/kg intravenous injection of 50B8-DP100 si-NPs silenced the tumor driver gene Rictor at the protein level by 80% in an orthotopic breast tumor model. 50B8-DP100 si-NPs delivering siRictor were assessed for therapeutic efficacy in an orthotopic HCC70 mammary tumor model. This formulation significantly inhibited tumor growth compared to siControl-NP treatment. 50B8-DP100 si-NPs were also evaluated for safety and were well-tolerated following a multi-dose treatment scheme. This work provides new insight on ternary si-NP structure-function relationships and identifies core polymer optimization strategies that can yield safe si-NP formulations with potent oncogene silencing.

Intraocular Sustained Release of EPO-R76E Mitigates Glaucoma Pathogenesis by Activating the NRF2/ARE Pathway.

Erythropoietin (EPO) is neuroprotective in multiple models of neurodegenerative diseases, including glaucoma. EPO-R76E retains the neuroprotective effects of EPO but diminishes the effects on hematocrit. Treatment with EPO-R76E in a glaucoma model increases expression of antioxidant proteins and is neuroprotective. A major pathway that controls the expression of antioxidant proteins is the NRF2/ARE pathway. This pathway is activated endogenously after elevation of intraocular pressure (IOP) and contributes to the slow onset of pathology in glaucoma. In this study, we explored if sustained release of EPO-R76E in the eye would activate the NRF2/ARE pathway and if this pathway was key to its neuroprotective activity. Treatment with PLGA.EPO-E76E prevented increases in retinal superoxide levels in vivo, and caused phosphorylation of NRF2 and upregulation of antioxidants. Further, EPO-R76E activates NRF2 via phosphorylation by the MAPK pathway rather than the PI3K/Akt pathway, used by the endogenous antioxidant response to elevated IOP.

Structural Optimization of siRNA Conjugates for Albumin Binding Achieves Effective MCL1-Targeted Cancer Therapy.

The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.

A Reactive Oxygen Species-Scavenging 'Stealth' Polymer, Poly(thioglycidyl glycerol), Outperforms Poly(ethylene glycol) in Protein Conjugates and Nanocarriers and Enhances Protein Stability to Environmental and Biological Stressors.

This study addresses well-known shortcomings of poly(ethylene glycol) (PEG)-based conjugates. PEGylation is by far the most common method employed to overcome immunogenicity and suboptimal pharmacokinetics of, for example, therapeutic proteins but has significant drawbacks. First, PEG offers no protection from denaturation during lyophilization, storage, or oxidation (e.g., by biological oxidants, reactive oxygen species); second, PEG's inherent immunogenicity, leading to hypersensitivity and accelerated blood clearance (ABC), is a growing concern. We have here developed an 'active-stealth' polymer, poly(thioglycidyl glycerol)(PTGG), which in human plasma is less immunogenic than PEG (35% less complement activation) and features a reactive oxygen species-scavenging and anti-inflammatory action (∼50% less TNF-α in LPS-stimulated macrophages at only 0.1 mg/mL). PTGG was conjugated to proteins via a one-pot process; molar mass- and grafting density-matched PTGG-lysozyme conjugates were superior to their PEG analogues in terms of enzyme activity and stability against freeze-drying or oxidation; the latter is due to sacrificial oxidation of methionine-mimetic PTGG chains. Both in mice and rats, PTGG-ovalbumin displayed circulation half-lives up to twice as long as PEG-ovalbumin, but most importantly─and differently from PEG─without any associated ABC effect seen either in the time dependency of blood concentration, in the liver/splenic accumulation, or in antipolymer IgM/IgG titers. Furthermore, similar pharmacokinetic results were obtained with PTGGylated/PEGylated liposomal nanocarriers. PTGG's 'active-stealth' character therefore makes it a highly promising alternative to PEG for conjugation to biologics or nanocarriers.

Reactive oxygen species-degradable polythioketal urethane foam dressings to promote porcine skin wound repair.

Porous, resorbable biomaterials can serve as temporary scaffolds that support cell infiltration, tissue formation, and remodeling of nonhealing skin wounds. Synthetic biomaterials are less expensive to manufacture than biologic dressings and can achieve a broader range of physiochemical properties, but opportunities remain to tailor these materials for ideal host immune and regenerative responses. Polyesters are a well-established class of synthetic biomaterials; however, acidic degradation products released by their hydrolysis can cause poorly controlled autocatalytic degradation. Here, we systemically explored reactive oxygen species (ROS)-degradable polythioketal (PTK) urethane (UR) foams with varied hydrophilicity for skin wound healing. The most hydrophilic PTK-UR variant, with seven ethylene glycol (EG7) repeats flanking each side of a thioketal bond, exhibited the highest ROS reactivity and promoted optimal tissue infiltration, extracellular matrix (ECM) deposition, and reepithelialization in porcine skin wounds. EG7 induced lower foreign body response, greater recruitment of regenerative immune cell populations, and resolution of type 1 inflammation compared to more hydrophobic PTK-UR scaffolds. Porcine wounds treated with EG7 PTK-UR foams had greater ECM production, vascularization, and resolution of proinflammatory immune cells compared to polyester UR foam-based NovoSorb Biodegradable Temporizing Matrix (BTM)-treated wounds and greater early vascular perfusion and similar wound resurfacing relative to clinical gold standard Integra Bilayer Wound Matrix (BWM). In a porcine ischemic flap excisional wound model, EG7 PTK-UR treatment led to higher wound healing scores driven by lower inflammation and higher reepithelialization compared to NovoSorb BTM. PTK-UR foams warrant further investigation as synthetic biomaterials for wound healing applications.

Optimizing an Antioxidant TEMPO Copolymer for Reactive Oxygen Species Scavenging and Anti-Inflammatory Effects in Vivo.

Oxidative stress is broadly implicated in chronic, inflammatory diseases because it causes protein and lipid damage, cell death, and stimulation of inflammatory signaling. Supplementation of innate antioxidant mechanisms with drugs such as the superoxide dismutase (SOD) mimetic compound 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) is a promising strategy for reducing oxidative stress-driven pathologies. TEMPO is inexpensive to produce and has strong antioxidant activity, but it is limited as a drug due to rapid clearance from the body. It is also challenging to encapsulate into micellar nanoparticles or polymer microparticles, because it is a small, water soluble molecule that does not efficiently load into hydrophobic carrier systems. In this work, we pursued a polymeric form of TEMPO [poly(TEMPO)] to increase its molecular weight with the goal of improving in vivo bioavailability. High density of TEMPO on the poly(TEMPO) backbone limited water solubility and bioactivity of the product, a challenge that was overcome by tuning the density of TEMPO in the polymer by copolymerization with the hydrophilic monomer dimethylacrylamide (DMA). Using this strategy, we formed a series of poly(DMA-co-TEMPO) random copolymers. An optimal composition of 40 mol % TEMPO/60 mol % DMA was identified for water solubility and O2•- scavenging in vitro. In an air pouch model of acute local inflammation, the optimized copolymer outperformed both the free drug and a 100% poly(TEMPO) formulation in O2•- scavenging, retention, and reduction of TNFα levels. Additionally, the optimized copolymer reduced ROS levels after systemic injection in a footpad model of inflammation. These results demonstrate the benefit of polymerizing TEMPO for in vivo efficacy and could lead to a useful antioxidant polymer formulation for next-generation anti-inflammatory treatments.

Recent Advances in Clinical Translation of Intra-Articular Osteoarthritis Drug Delivery Systems.

Osteoarthritis (OA) is a degenerative disease of the joints and a leading cause of physical disability in adults. Intra-articular (IA) therapy is a popular treatment strategy for localized, single-joint OA; however, small-molecule drugs such as corticosteroids do not provide prolonged relief. One possible reason for their lack of efficacy is high clearance rates from the joint through constant lymphatic drainage of the synovial tissues and synovial fluid and also by their exchange via the synovial vasculature. Advanced drug delivery strategies for extended release of therapeutic agents in the joint space is a promising approach to improve outcomes for OA patients. Broadly, the basic principle behind this strategy is to encapsulate therapeutic agents in a polymeric drug delivery system (DDS) for diffusion- and/or degradation-controlled release, whereby degradation can occur by hydrolysis or tied to relevant microenvironmental cues such as pH, reactive oxygen species (ROS), and protease activity. In this review, we highlight the development of clinically tested IA therapies for OA and highlight recent systems which have been investigated preclinically. DDS strategies including hydrogels, liposomes, polymeric microparticles (MPs) and nanoparticles (NPs), drug conjugates, and combination systems are introduced and evaluated for clinical translational potential.

Novel approaches for the design, delivery and administration of vaccine technologies.

It is easy to argue that vaccine development represents humankind's most important and successful endeavour, such is the impact that vaccination has had on human morbidity and mortality over the last 200 years. During this time the original method of Jenner and Pasteur, i.e. that of injecting live-attenuated or inactivated pathogens, has been developed and supplemented with a wide range of alternative approaches which are now in clinical use or under development. These next-generation technologies have been designed to produce a vaccine that has the effectiveness of the original live-attenuated and inactivated vaccines, but without the associated risks and limitations. Indeed, the method of development has undoubtedly moved away from Pasteur's three Is paradigm (isolate, inactivate, inject) towards an approach of rational design, made possible by improved knowledge of the pathogen-host interaction and the mechanisms of the immune system. These novel vaccines have explored methods for targeted delivery of antigenic material, as well as for the control of release profiles, so that dosing regimens can be matched to the time-lines of immune system stimulation and the realities of health-care delivery in dispersed populations. The methods by which vaccines are administered are also the subject of intense research in the hope that needle and syringe dosing, with all its associated issues regarding risk of injury, cross-infection and patient compliance, can be replaced. This review provides a detailed overview of new vaccine vectors as well as information pertaining to the novel delivery platforms under development.

Demonstration of extended capture range for James Webb Space Telescope phase retrieval.

A geometrical phase retrieval (GPR) algorithm is applied to the problem of image stacking in order to extend the capture range of normal phase retrieval (PR) on the James Webb Space Telescope (JWST), and potentially eliminate a lengthy image-stacking process that is based on centroids. Computer simulations are used to establish the capture range of the existing PR algorithm for JWST and demonstrate that it is increased by more than a factor of 10 when combined with GPR, guaranteeing PR capture 95% of the time. An experiment using a scale optical model of JWST was conducted to demonstrate the effectiveness of the GPR algorithm in both coherent and incoherent imaging.

Ultrasound-induced inertial cavitation from gas-stabilizing nanoparticles.

The understanding of cavitation from nanoparticles has been hindered by the inability to control nanobubble size. We present a method to manufacture nanoparticles with a tunable single hemispherical depression (nanocups) of mean diameter 90, 260, or 650 nm entrapping a nanobubble. A modified Rayleigh-Plesset crevice model predicts the inertial cavitation threshold as a function of cavity size and frequency, and is verified experimentally. The ability to tune cavitation nanonuclei and predict their behavior will be useful for applications ranging from cancer therapy to ultrasonic cleaning.

Combining virotherapy and angiotherapy for the treatment of breast cancer.

A breast cancer-selective oncolytic adenovirus was engineered to express antagonists of vascular endothelial growth factor (VEGF) and Notch signaling to combine direct anticancer activity with disruption of tumor-associated angiogenesis. Replication of the parental virus, AdEHE2F, is stimulated by estrogen receptor (ER), E2F1 and hypoxia, and it mediates selective lysis of breast cancer cells in vitro and in vivo. Here, we encoded soluble Flt-1 (sFlt1) and soluble Dll4 (sDll4) under control of the E3 promoter. sFlt1 (the extra-cellular domain of VEGF receptor 1) binds VEGF-A and inhibits stimulation of VEGFR2, decreasing angiogenic stimulus. Conversely, sDll4 (the extracellular domain of Delta-like 4) antagonizes Notch signaling to prevent endothelial maturation. We hypothesized that these agents might show additive or synergistic activity. In vitro, sFlt1 inhibited endothelial cell proliferation and sprouting, whereas sDll4 increased the number of vascular branchpoints. In ER-positive ZR75.1 tumors in vivo AdEHE2F showed the potent direct virotherapy with no augmentation owing to sFlt1 or sDll4; however, in ER-negative MDA-231 tumors efficacy was enhanced by encoding sFlt1 or sDll4, with survival time extending to double that of controls. There was also a dramatic decrease in the total number of tumour blood vessels, as well as the number of perfused vessels, suggesting that improved efficacy reflects combined anti-tumour and anti-vascular effects.

Comparison of molecular strategies for breast cancer virotherapy using oncolytic adenovirus.

Oncolytic viruses are regulated by the tumor phenotype to replicate and lyse cancer cells selectively. To identify optimal strategies for breast cancer we compared five adenoviruses with distinct regulatory mechanisms: Ad-dl922-947 (targets G1-S checkpoint); Ad-Onyx-015 and Ad-Onyx-017 (target p53/mRNA export); Ad-vKH1 (targets Wnt pathway), and AdEHE2F (targets estrogen receptor/G1-S checkpoint/hypoxic signaling). The quantity of virus required to kill 50% of breast cancer cells after 6 days (EC(50), plaque-forming units per cell) was measured. The most potent virus was Ad-dl922-947 (EC(50), 0.01-5.4 in SkBr3, MDA-231, MDA-468, MCF7, and ZR75.1 cells), followed by wild-type (Ad-WT; EC(50), 0.3-5.5) and AdEHE2F (EC(50), 1.4-3.9). Ad-vKH1 (EC(50), 7.2-72.1), Ad-Onyx-017 (EC(50), 8.4-167), and Ad-Onyx-015 (EC(50), 17.7-377) showed less activity. Most viruses showed limited cytotoxicity in normal human cells, including breast epithelium MCF10A (EC(50), >722) and fibroblasts (EC(50), >192) and only moderate cytotoxicity in normal microvascular endothelial cells (HMVECs; EC(50), 42.8-149), except Ad-dl922-947, which was active in HMVECs (EC(50), 1.6). After injection into MDA-231 xenografts, Ad-WT, AdEHE2F, and Ad-dl922-947 showed replication, assessed by hexon staining and quantitative polymerase chain reaction measurement of viral DNA, and significantly inhibited tumor growth, leading to extended survival. After intravenous injection Ad-dl922-947 showed DNA replication (233% of the injected dose was measured in liver after 3 days) whereas AdEHE2F did not. Overall, AdEHE2F showed the best combination of low toxicity in normal cells and high activity in breast cancer in vitro and in vivo, suggesting that molecular targeting using estrogen response elements, hypoxia response elements, and a dysregulated G1-S checkpoint is a promising strategy for virotherapy of breast cancer.

Factors influencing retention of adenovirus within tumours following direct intratumoural injection.

Direct intratumoural (IT) administration of adenovirus is widely used, however little is known about the resulting distribution of virus particles. Here we have evaluated the influence of tumour size, volume of injectate and occlusion of injection sites (to prevent retrograde seepage) on particle biodistribution and transgene expression. In subcutaneous MDA-231 xenografts, IT injection of relatively large volumes (4 x 20% (vol/vol) injections) resulted in just 40% of the administered dose being retained in tumour tissue after 30 min, with 15% in the liver thought to reflect systemic 'overflow'. Occlusion of the injection sites using surgical adhesive increased retention of the vector to 80% in the tumour with no increase in liver levels. Spread of expression was enhanced using multiple injection sites, but not by using larger injectate volumes. In ZR75.1 breast carcinoma xenografts virus distribution was different, with no evidence of systemic overflow leading to hepatic transduction following IT injection. Typically, clinical doses employ up to 30% vol/vol IT injections. Depending on the tumour, this may give considerable systemic overflow and might account for the high frequency of fevers observed. Virus performance might be improved by tailoring volumes and frequency of IT injection for tumour biology or histotype.

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 Ad(GFP)DNA bearing TP (Ad(GFP)DNA-TP), delivered with DOTAP, gave a two-fold greater frequency of transduction than Ad(GFP)DNA(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/Ad(wild type)DNA-TP to mediate a powerful cytopathic effect. Similarly in vivo, direct injection of a polyplex/Ad(wild type)DNA-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.

Changing epidemiology of hepatitis A: should we be doing more to vaccinate injecting drug users?

Since 2001 there have been significant outbreaks of hepatitis A virus (HAV) across South Yorkshire, largely in intravenous drug users, and HAV infection has been reported to be an increasing problem in England and Scotland during this time. This paper reports a brief investigation to clarify current HAV epidemiology in England and Wales. The epidemiology of HAV in England, but not yet Wales, has recently changed. Laboratory reports now show that most cases are occurring in young adults, mainly young men, and that the commonest reported risk group is injecting drug users. That cases may now be concentrated in injecting drug users is supported by reports from consultants in communicable disease control (CsCDC). These detail fourteen outbreaks in England in 2002 alone, all involving injecting drug users. Links to prisons and to the homeless, usually those in hostels, were also common. A combined Hepatitis A/B vaccine is readily available and we recommend that this now be used to extend the national immunisation programme against Hepatitis B in injecting drug users to include HAV.