Christopher T. Turner

Analysis of normal and mutant iduronate-2-sulphatase conformation.

Mammalian sulphatases (EC 3.1.6) are a family of enzymes that have a high degree of similarity in amino acid sequence, structure and catalytic mechanism. IDS (iduronate-2-sulphatase; EC 3.1.6.13) is a lysosomal exo-sulphatase that belongs to this protein family and is involved in the degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate. An IDS deficiency causes the lysosomal storage disorder MPS II (mucopolysaccharidosis type II). To examine the structural alterations in heat-denatured and mutant IDS, a panel of four monoclonal antibodies was raised to the denatured protein and used as probes of protein conformation. The linear sequence epitope reactivity of a polyclonal antibody raised against the native protein and the monoclonal antibodies were defined and mapped to distinct regions on the IDS protein. The antigenicity of native IDS was higher in regions without glycosylation, but reactivity was not restricted to protein surface epitopes. One monoclonal epitope was relatively surface accessible and in close proximity to an N-linked glycosylation site, while three others required additional thermal energy to expose the epitopes. The monoclonal antibodies demonstrated the capacity to differentiate progressive structural changes in IDS and could be used to characterize the severity of MPS type II in patients based on variable denatured microstates.

Surface engineering of porous silicon to optimise therapeutic antibody loading and release

The proinflammatory cytokine, tumor necrosis factor-α (TNF-α), is elevated in several diseases such as uveitis, rheumatoid arthritis and non-healing chronic wounds. Adding Infliximab, a chimeric IgG1 monoclonal antibody raised against TNF-α, to chronic wound fluid can neutralise human TNF-α, thereby providing a potential therapeutic option for chronic wound healing. However, to avoid the need for repeated application in a clinical setting, and to protect the therapeutic antibody from the hostile environment of the wound, suitable delivery vehicles are required. Porous silicon (pSi) is a biodegradable high surface area material commonly employed for drug delivery applications. In this study, the use of pSi microparticles (pSi MPs) for the controlled release of Infliximab to disease environments, such as chronic wounds, is demonstrated. Surface chemistry and pore parameters for Infliximab loading are first optimised in pSi films and loading conditions are transferred to pSi MPs. Loading regimens exceeding 60 μg of Infliximab per mg of pSi are achieved. Infliximab is released with zero-order release kinetics over the course of 8 days. Critically, the released antibody remains functional and is able to sequester TNF-α over a weeklong timeframe; suitable for a clinical application in chronic wound therapy.

Delivery of Flightless I Neutralizing Antibody from Porous Silicon Nanoparticles Improves Wound Healing in Diabetic Mice

&NA; Flightless I (Flii) is elevated in human chronic wounds and is a negative regulator of wound repair. Decreasing its activity improves healing responses. Flii neutralizing antibodies (FnAbs) decrease Flii activity in vivo and hold significant promise as healing agents. However, to avoid the need for repeated application in a clinical setting and to protect the therapeutic antibody from the hostile environment of the wound, suitable delivery vehicles are required. In this study, the use of porous silicon nanoparticles (pSi NPs) is demonstrated for the controlled release of FnAb to diabetic wounds. We achieve FnAb loading regimens exceeding 250 µg antibody per mg of vehicle. FnAb‐loaded pSi NPs increase keratinocyte proliferation and enhance migration in scratch wound assays. Release studies confirm the functionality of the FnAb in terms of Flii binding. Using a streptozotocin‐induced model of diabetic wound healing, a significant improvement in healing is observed for mice treated with FnAb‐loaded pSi NPs compared to controls, including FnAb alone. FnAb‐loaded pSi NPs treated with proteases show intact and functional antibody for up to 7 d post‐treatment, suggesting protection of the antibodies from proteolytic degradation in wound fluid. pSi NPs may therefore enable new therapeutic approaches for the treatment of diabetic ulcers. &NA; Flightless neutralizing antibody (FnAb) is packaged into porous silicon nanoparticles (pSi NPs), protecting the drug from the harsh diabetic wound environment. pSi NPs have a high antibody‐loading capacity, extend antibody release, and critically retain antibody functionality. The application of FnAb‐loaded pSi NPs to diabetic wounds in mice leads to significant improvements in healing compared to wounds treated with FnAb alone. Figure. No caption available.

Therapeutic potential of inorganic nanoparticles for the delivery of monoclonal antibodies

Monoclonal antibodies (mAbs), available for a range of diseases, including tumours, leukemia, and multiple sclerosis, are emerging as the fastest growing area of therapeutic drug development. The greatest advantage of therapeutic mAbs is their ability to bind with a high degree of specificity to target proteins involved in disease pathophysiology. In response, effector functions are triggered and these ameliorate the disease cascade. As an alternative to this reliance on effector functions, drugs can be conjugated to mAbs. The ability to target compounds to the site of pathology minimises the nonspecific side effects associated with systemic administration. In both instances, optimising the delivery, absorption, and distribution of the mAbs, whilst minimising potential side effects, remain the key hurdles to improved clinical outcomes. Novel delivery strategies are being investigated with more vigour in recent years, and nanoparticles are being identified as suitable vehicles. In conjunction with permitting a controlled release profile, nanoparticles protect the drug from degradation, reducing both the dose and frequency of administration. Moreover, these particles shield the patient from the immune complications associated with high dose mAb infusions or drug cytotoxicity. This review outlines recent advances in nanoparticle technology and how they may be of benefit as therapeutic mAb delivery/targeting vehicles.

Immune response to enzyme replacement therapy: single epitope control of antigen distribution from circulation.

Immune response to replacement therapy has been reported for a range of therapeutic strategies being developed for the treatment of patients with genetic disease. The potential problem of immune response to enzyme replacement therapy has been investigated in alpha-L-iduronidase immunized rats, representing a model of the lysosomal storage disorder Hurler syndrome (alpha-L-iduronidase deficiency). The antibody response to alpha-L-iduronidase showed that the positional location of antibody reactivity was similar for different immunized rats, but the precise linear sequence epitopes identified, varied between rats. A monoclonal antibody reacting to an epitope in close proximity to one high antigenicity site on alpha-L-iduronidase was used to reproduce the in vivo effect of altered enzyme tissue distribution, previously observed in immunized rats infused with alpha-L-iduronidase. The study demonstrated that during an immune response, antibody reacting to a single epitope could partially control the tissue distribution of antigen from circulation.

Fibroblast-specific upregulation of Flightless I impairs wound healing

The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell‐specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue‐specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast‐specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue‐specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non‐inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non‐tissue‐specific upregulation of Flii suggesting that fibroblast‐derived Flii may have an important role in the wound healing response.

Flightless I is a key regulator of the fibroproliferative process in hypertrophic scarring and a target for a novel antiscarring therapy

Hypertrophic scarring carries a large burden of disease, including disfigurement, pain and disability. There is currently no effective medical treatment to reduce or prevent hypertrophic scarring. Flightless I (Flii), a member of the gelsolin family of actin remodelling proteins, is an important negative regulator of wound repair.

Drug induced exocytosis of glycogen in Pompe disease

Pompe disease is caused by a deficiency in the lysosomal enzyme α-glucosidase, and this leads to glycogen accumulation in the autolysosomes of patient cells. Glycogen storage material is exocytosed at a basal rate in cultured Pompe cells, with one study showing up to 80% is released under specific culture conditions. Critically, exocytosis induction may reduce glycogen storage in Pompe patients, providing the basis for a therapeutic strategy whereby stored glycogen is redirected to an extracellular location and subsequently degraded by circulating amylases. The focus of the current study was to identify compounds capable of inducing rapid glycogen exocytosis in cultured Pompe cells. Here, calcimycin, lysophosphatidylcholine and α-l-iduronidase each significantly increased glycogen exocytosis compared to vehicle-treated controls. The most effective compound, calcimycin, induced exocytosis through a Ca2+-dependent mechanism, although was unable to release a pool of vesicular glycogen larger than the calcimycin-induced exocytic pore. There was reduced glycogen release from Pompe compared to unaffected cells, primarily due to increased granule size in Pompe cells. Drug induced exocytosis therefore shows promise as a therapeutic approach for Pompe patients but strategies are required to enhance the release of large molecular weight glycogen granules.

Mass spectrometric quantification of glycogen to assess primary substrate accumulation in the Pompe mouse.

Glycogen storage in the α-glucosidase knockout((6neo/6neo)) mouse recapitulates the biochemical defect that occurs in the human condition; as such, this mouse serves as a model for the inherited metabolic deficiency of lysosomal acid α-glucosidase known as Pompe disease. Although this model has been widely used for the assessment of therapies, the time course of glycogen accumulation that occurs as untreated Pompe mice age has not been reported. To address this, we developed a quantitative method involving amyloglucosidase digestion of glycogen and quantification of the resulting free glucose by liquid chromatography/electrospray ionization-tandem mass spectrometry. The method was sensitive enough to measure as little as 0.1 μg of glycogen in tissue extracts with intra- and interassay coefficients of variation of less than 12%. Quantification of glycogen in tissues from Pompe mice from birth to 26 weeks of age showed that, in addition to the accumulation of glycogen in the heart and skeletal muscle, glycogen also progressively accumulated in the brain, diaphragm, and skin. Glycogen storage was also evident at birth in these tissues. This method may be particularly useful for longitudinal assessment of glycogen reduction in response to experimental therapies being trialed in this model.

Glycogen Exocytosis from Cultured Pompe Skin Fibroblasts

Objective: Pompe disease is a progressive form of muscular dystrophy caused by a deficiency in the lysosomal enzyme α-glucosidase (GAA), and leads to the accumulation of glycogen in affected cells. Enzyme replacement therapy is approved to treat infantile-onset Pompe disease, but this is not completely effective, necessitating the development of new therapeutic strategies. Exocytosis involves the fusion of intracellular vesicles with the cell surface and the release of vesicular content, and is a mechanism that could be used in Pompe disease to remove stored glycogen from affected cells. The exocytosis of storage material from Pompe patient cells into circulation could result in glycogen degradation by other amylases (i.e. not GAA) and this could be developed in the future as a new or adjunct therapeutic strategy. Methods: A sensitive mass spectrometry assay was used to quantify glycogen in cell extracts and the culture media from confluent Pompe skin fibroblasts. Results: Four percent of vesicular glycogen was exocytosed after 2 hours in culture. This natural process of glycogen exocytosis was enhanced in sub-confluent Pompe cells, which released >80% of glycogen after 2 hours in culture. Conclusion: Under appropriate conditions exocytosis can release most of the stored glycogen in Pompe skin fibroblasts, identifying a potential target for therapeutic intervention.