Spray drying: Inhalable powders for pulmonary gene therapy.

Gene therapy holds potential in the treatment of many lung pathologies, as indicated by the growing number of clinical trials in recent decades. Pulmonary delivery of gene therapies via inhalation enables localised treatment while the extensive lung surface area promotes enhanced drug absorption. However, loss of nucleic acid integrity during the aerosolisation process, pulmonary clearance, and undesirable drug deposition, pose a major challenge for local delivery. Therefore, the development of nucleic acids into a stable inhalable pharmaceutical preparation would be advantageous. Dry powder inhalers (DPIs) are considered superior compared to nebulisation and pressurised-metered dose inhalers (pMDIs) due to the production of a stable dry formulation, an easy dispensing process, and minimal physical stress. DPIs are commonly produced via spray drying with a range of excipients, solvents, and separation options which can be modified to improve the stability of the nucleic acid cargo. This review details the ideal characteristics for pulmonary delivery and formulation of DPIs for gene therapy to the lungs. The utilisation of spray drying for the production of nucleic acid-containing DPIs is evaluated, with a specific focus on the influence of instrument parameters, the nucleic acid delivery system, and excipients with respect to cargo stability and functionality.

Design of a novel electrospun PVA platform for gene therapy applications using the CHAT peptide.

The electrospinning of polymers has previously shown excellent potential for localised gene therapy. Thus, it was proposed that for the first time, the cell-penetrating CHAT peptide could be utilised to deliver DNA via electrospun nanofibres for localised gene therapy treatment. CHAT is an effective delivery system that encapsulates pDNA to form nanoparticles with the physicochemical characteristics for cellular uptake and protein generation. In this study, the production of smooth, bead-free PVA nanofibres by electrospinning was optimised through a Design of Experiments approach. Bead-free PVA nanofibres were consistently produced using the optimised parameters as follows: applied voltage (8 kV); collector-emitter distance (8 cm); polymer flow rate (4 µL/min); polymer concentration (9 wt% polymer); PVA MW (146-180 kDa). PVA nanofibres were subsequently crosslinked in 1 vol% glutaraldehyde in methanol to confer stability under aqueous conditions with minimal change to morphology, and no compromise to biocompatibility. Nanoparticles of CHAT/pDNA were synthesised and incorporated into the crosslinked nanofibres via soak-loading. Evaluation studies indicated that 100% of the loaded cargo was released within 48 h from the nanofibres. Furthermore, the released pDNA retained structural integrity and functionality as confirmed by gel electrophoresis and transfection studies in NCTC-929 fibroblast cells. Taken together, this data demonstrates that delivery of CHAT/pDNA nanoparticles from electrospun PVA nanofibres represents a solution for localised gene therapy.

Pore-forming bioinks to enable spatio-temporally defined gene delivery in bioprinted tissues.

The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-ß3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.

Influence of alginate backbone on efficacy of thermo-responsive alginate-g-P(NIPAAm) hydrogel as a vehicle for sustained and controlled gene delivery.

Alginate grafted poly(N-isopropylacrylamide) hydrogels (Alg-g-P(NIPAAm)) form three-dimensional networks in mild conditions, making them suitable for incorporation of labile macromolecules, such as DNA. The impact of P(NIPAAm) on copolymer characteristics has been well studied, however the impact of alginate backbone characteristics on copolymer properties has to-date not been investigated. Six different Alg-g-P(NIPAAm) hydrogels were synthesised with 10% alginate, which varied in terms of molecular weight (MW) and mannuronate/guluronate (M/G) monomer ratio, and with 90% NIPAAm in order to develop an injectable and thermo-responsive hydrogel formulation for localised gene delivery. Hydrogel stiffness was directly proportional to MW and the M/G ratio of the alginate backbone. Hydrogels with a high MW or low M/G ratio alginate backbone demonstrated a greater stiffness than those hydrogels comprising low MW alginates and high M/G ratio. Hydrogels with a high M/G ratio also produced a complexed and meshed hydrogel network while hydrogels with a low M/G ratio produced a simplified structure with the superposition of Alg-g-P(NIPAAm) sheets. This study was designed to produce the optimal Alg-g-P(NIPAAm) hydrogel with respect to localised delivery of DNA nanoparticles as a potential medical device for those with castrate resistant prostate cancer (CRPC). Given that CRPC typically disseminates to bone causing pain, morbidity and a plethora of skeletal related events, a copolymer based hydrogel was designed to for long term release of therapeutic DNA nanoparticles. The nanoparticles were comprised of plasmid DNA (pDNA), complexed with an amphipathic cell penetrating peptide termed RALA that is designed to enter cells with high efficiency. Alginate MW and M/G ratio affected stiffness, structure, injectability and degradation of the Alg-g-P(NIPAAm) hydrogel. Algogel 3001, had the optimal characteristics for long-term application and was loaded with RALA/pDNA NPs. From the release profiles, it was evident that RALA protected the pDNA from degradation over a 30-day period and conferred a sustained and controlled release profile from the hydrogels compared to pDNA only. Taken together, we have designed a slowly degrading hydrogel suitable for sustained delivery of nucleic acids when incorporated with the RALA delivery peptide. This now opens up several opportunities for the delivery of therapeutic pDNA from this thermo-responsive hydrogel with numerous medical applications.

Mesenchymal stem cell fate following non-viral gene transfection strongly depends on the choice of delivery vector.

Controlling the phenotype of mesenchymal stem cells (MSCs) through the delivery of regulatory genes is a promising strategy in tissue engineering (TE). Essential to effective gene delivery is the choice of gene carrier. Non-viral delivery vectors have been extensively used in TE, however their intrinsic effects on MSC differentiation remain poorly understood. The objective of this study was to investigate the influence of three different classes of non-viral gene delivery vectors: (1) cationic polymers (polyethylenimine, PEI), (2) inorganic nanoparticles (nanohydroxyapatite, nHA) and (3) amphipathic peptides (RALA peptide) on modulating stem cell fate after reporter and therapeutic gene delivery. Despite facilitating similar reporter gene transfection efficiencies, these nanoparticle-based vectors had dramatically different effects on MSC viability, cytoskeletal morphology and differentiation. After reporter gene delivery (pGFP or pLUC), the nHA and RALA vectors supported an elongated MSC morphology, actin stress fibre formation and the development of mature focal adhesions, while cells appeared rounded and less tense following PEI transfection. These changes in MSC morphology correlated with enhanced osteogenesis following nHA and RALA transfection and adipogenesis following PEI transfection. When therapeutic genes encoding for transforming growth factor beta 3 (TGF-ß3) and/or bone morphogenic protein 2 (BMP2) were delivered to MSCs, nHA promoted osteogenesis in 2D culture and the development of an endochondral phenotype in 3D culture, while RALA was less osteogenic and appeared to promote a more stable hyaline cartilage-like phenotype. In contrast, PEI failed to induce robust osteogenesis or chondrogenesis of MSCs, despite effective therapeutic protein production. Taken together, these results demonstrate that the differentiation of MSCs through the application of non-viral gene delivery strategies depends not only on the gene delivered, but also on the gene carrier itself. STATEMENT OF SIGNIFICANCE: Nanoparticle-based non-viral gene delivery vectors have been extensively used in regenerative medicine, however their intrinsic effects on mesenchymal stem cell (MSC) differentiation remain poorly understood. This paper demonstrates that different classes of commonly used non-viral vectors are not inert and they have a strong effect on cell morphology, stress fiber formation and gene transcription in MSCs, which in turn modulates their capacity to differentiate towards osteogenic, adipogenic and chondrogenic lineages. These results also point to the need for careful and tissue-specific selection of nanoparticle-based delivery vectors to prevent undesired phenotypic changes and off-target effects when delivering therapeutic genes to damaged or diseased tissues.

Critical review: Injectability of calcium phosphate pastes and cements.

Calcium phosphate cements (CPC) have seen clinical success in many dental and orthopaedic applications in recent years. The properties of CPC essential for clinical success are reviewed in this article, which includes properties of the set cement (e.g. bioresorbability, biocompatibility, porosity and mechanical properties) and unset cement (e.g. setting time, cohesion, flow properties and ease of delivery to the surgical site). Emphasis is on the delivery of calcium phosphate (CaP) pastes and CPC, in particular the occurrence of separation of the liquid and solid components of the pastes and cements during injection; and established methods to reduce this phase separation. In addition a review of phase separation mechanisms observed during the extrusion of other biphasic paste systems and the theoretical models used to describe these mechanisms are discussed. STATEMENT OF SIGNIFICANCE: Occurrence of phase separation of calcium phosphate pastes and cements during injection limits their full exploitation as a bone substitute in minimally invasive surgical applications. Due to lack of theoretical understanding of the phase separation mechanism(s), optimisation of an injectable CPC that satisfies clinical requirements has proven difficult. However, phase separation of pastes during delivery has been the focus across several research fields. Therefore in addition to a review of methods to reduce phase separation of CPC and the associated constraints, a review of phase separation mechanisms observed during extrusion of other pastes and the theoretical models used to describe these mechanisms is presented. It is anticipated this review will benefit future attempts to develop injectable calcium phosphate based systems.

Identification of RBCK1 as a novel regulator of FKBPL: implications for tumor growth and response to tamoxifen.

FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identified a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-ß-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-α (ERα). Furthermore, FKBPL and RBCK1 associate with ERα at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.

The tissue plasminogen activator gene promoter: a novel tool for radiogenic gene therapy of the prostate?

BACKGROUND: Radiation therapy is a treatment modality routinely used in cancer management so it is not unexpected that radiation-inducible promoters have emerged as an attractive tool for controlled gene therapy. The human tissue plasminogen activator gene promoter (t-PA) has been proposed as a candidate for radiogenic gene therapy, but has not been exploited to date. The purpose of this study was to evaluate the potential of this promoter to drive the expression of a reporter gene, the green fluorescent protein (GFP), in response to radiation exposure. METHODS: To investigate whether the promoter could be used for prostate cancer gene therapy, we initially transfected normal and malignant prostate cells. We then transfected HMEC-1 endothelial cells and ex vivo rat tail artery and monitored GFP levels using Western blotting following the delivery of single doses of ionizing radiation (2, 4, 6 Gy) to test whether the promoter could be used for vascular targeted gene therapy. RESULTS: The t-PA promoter induced GFP expression up to 6-fold in all cell types tested in response to radiation doses within the clinical range. CONCLUSIONS: These results suggest that the t-PA promoter may be incorporated into gene therapy strategies driving therapeutic transgenes in conjunction with radiation therapy.

Nitric oxide--a novel therapeutic for cancer.

Much research over the past two decades has focussed on understanding the complex interactions of nitric oxide (NO(.)) in both physiological and pathological processes. As with many other aspects of NO(.) biology, its precise role in tumour pathophysiology has been the cause of intense debate and we now know that it participates in numerous signalling pathways that are crucial to the malignant character of cancer. The available experimental evidence highlights contrasting pro- and anti-tumour effects of NO(.) expression, which appear to be reconciled by consideration of the concentrations involved. This review addresses the complexities of the role of NO(.) in cancer, whilst evaluating various experimental approaches to NO(.)-based cancer therapies, including both inhibition of nitric oxide synthases, and overexpression of NO(.) using donor drugs or nitric oxide synthase gene transfer. The evidence provided strongly supports a role for manipulation of tumour NO(.) either as a stand-alone therapy or in combination with conventional treatments to achieve a significant therapeutic gain.

The radiation-inducible pE9 promoter driving inducible nitric oxide synthase radiosensitizes hypoxic tumour cells to radiation.

Driving high-level transgene expression in a tumour-specific manner remains a key requirement in the development of cancer gene therapy. We have previously demonstrated the strong anticancer effects of generating abnormally high levels of intracellular NO(*) following the overexpression of the inducible nitric oxide synthase (iNOS) gene. Much of this work has focused on utilizing exogenously activated promoters, which have been primarily induced using X-ray radiation. Here we further examine the potential of the pE9 promoter, comprising a combination of nine CArG radio-responsive elements, to drive the iNOS transgene. Effects of X-ray irradiation on promoter activity were compared in vitro under normoxic conditions and various degrees of hypoxia. The pE9 promoter generated high-level transgene expression, comparable with that achieved using the constitutively driven cytomegalovirus promoter. Furthermore, the radio-resistance of radiation-induced fibrosarcoma-1 (RIF-1) mouse sarcoma cells exposed to 0.1 and 0.01% O(2) was effectively eliminated following transfection with the pE9/iNOS construct. Significant inhibition of tumour growth was also observed in vivo following direct intratumoural injection of the pE9/iNOS construct compared to empty vector alone (P<0.001) or to a single radiation dose of 10 Gy (P<0.01). The combination of both therapies resulted in a significant 4.25 day growth delay compared to the gene therapy treatment alone (P<0.001). In summary, we have demonstrated the potential of the pE9/iNOS construct for reducing radio-resistance conferred by tumour cell hypoxia in vitro and in vivo, with greater tumour growth delay observed following the treatment with the gene therapy construct as compared with radiotherapy alone.

p21((WAF1))-mediated transcriptional targeting of inducible nitric oxide synthase gene therapy sensitizes tumours to fractionated radiotherapy.

Cancer gene therapy that utilizes toxic transgene products requires strict transcriptional targeting to prevent adverse normal tissue effects. We report on the use of a promoter derived from the cyclin dependent kinase inhibitor, p21((WAF1)), to control transgene expression. We demonstrate that this promoter is relatively silent in normal cells (L132, FSK, HMEC-1) compared to the almost constitutive expression obtained in tumour cells (DU145, LNCaP, HT29 and MCF-7) of varying p53 status, a characteristic that will be important in gene therapy protocols. In addition, we found that the p21((WAF1)) promoter could be further induced by both external beam radiation (up to eight-fold in DU145 cells), intracellular-concentrated radionuclides ([(211)At]MABG) (up to 3.5-fold in SK-N-BE(2c) cells) and hypoxia (up to four-fold in DU145 cells). We have previously achieved significant radiosensitization of tumour cells both in vitro and in vivo by using inducible nitric oxide synthase (iNOS) gene therapy to generate the potent radiosensitizer, nitric oxide (NO(.-)). Here, we report that a clinically relevant schedule of p21((WAF1))-driven iNOS gene therapy significantly sensitized both p53 wild-type RIF-1 tumours and p53 mutant HT29 tumours to fractionated radiotherapy. Our data highlight the utility of this p21((WAF1))/iNOS-targeted approach.

Tumor-selective drug activation: a GDEPT approach utilizing cytochrome P450 1A1 and AQ4N.

Drug metabolizing transgene products, which activate bioreductive cytotoxins, can be used to target treatment-resistant hypoxic tumors. The prodrug AQ4N is bioreduced in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Previously we have shown that intra-tumoral injection of CYP3A4 and CYP2B6 transgenes with AQ4N and radiation inhibits tumor growth. Here we examine the ability of other CYPs, in particular CYP1A1, to metabolize AQ4N, and to enhance radiosensitization. Metabolism of AQ4N was assessed using microsomes prepared from baculovirus-infected cells transfected with various CYP isoforms. AQ4N metabolism was most efficient with CYP1A1 (66.7 nmol/min/pmol) and 2B6 (34.4 nmol/min/pmol). Transient transfection of human CYP1A1+/-CYP reductase (CYPRED) was investigated in hypoxic RIF-1 mouse cells in vitro using the alkaline comet assay. There was a significant increase in DNA damage following transient transfection of CYP1A1 compared to non-transfected cells; inclusion of CYPRED provided no additional effect. In vivo, a single intra-tumoral injection of a CYP1A1 construct in combination with AQ4N (100 mg/kg i.p.) and 20 Gy X-rays caused a 16-day delay in tumor regrowth compared to tumors receiving AQ4N plus radiation and empty vector (P=0.0344). The results show the efficacy of a CYP1A1-mediated GDEPT strategy for bioreduction of AQ4N.

Parasite alteration of host shape: a quantitative approach to gigantism helps elucidate evolutionary advantages.

This investigation quantifies some aspects of the parasite-host relationship between the digenean Microphallus piriformes and its intermediate host Littorina saxatilis, the rough periwinkle. M. piriformes has an abridged life-cycle with no free-living stages, metacercariae remain within host viscera. Noticeable differences in shell shape of parasitized and uninfected periwinkles were investigated. These differences in shell shape were defined by growth parameters of height, diameter and beta angle. The relationship between these parameters was examined together with their impact on parasite reproduction. All 3 shape parameters were altered in periwinkles infected by M. piriformes. The alteration in beta angle and height increased the available volume for parasites in the shell spire by about 12%. As metacercarial production per sporocyst has been shown to depend on host size, the increased volume enables considerable additional life-time reproduction by the parasite, of approximately 550-850 additional metacercariae in hosts of the usual size range. The form of gigantism found in this study is discussed in relation to previous concepts. It is suggested that gigantism in permanently castrated hosts is adaptive parasite manipulation of host physiology, favoured in parasites with abbreviated life-cycles, when host viability increases parasite transmission, and when an initially small host individual is infected.