Neurotrophin gene augmentation by electrotransfer to improve cochlear implant hearing outcomes

This Review outlines the development of DNA-based therapeutics for treatment of hearing loss, and in particular, considers the potential to utilize the properties of recombinant neurotrophins to improve cochlear auditory (spiral ganglion) neuron survival and repair. This potential to reduce spiral ganglion neuron death and indeed re-grow the auditory nerve fibres has been the subject of considerable pre-clinical evaluation over decades with the view of improving the neural interface with cochlear implants. This provides the context for discussion about the development of a novel means of using cochlear implant electrode arrays for gene electrotransfer. Mesenchymal cells which line the cochlear perilymphatic compartment can be selectively transfected with (naked) plasmid DNA using array - based gene electrotransfer, termed ‘close-field electroporation’. This technology is able to drive expression of brain derived neurotrophic factor (BDNF) in the deafened guinea pig model, causing re-growth of the spiral ganglion peripheral neurites towards the mesenchymla cells, and hence into close proximity with cochlear implant electrodes within scala tympani. This was associated with functional enhancement of the cochlear implant neural interface (lower neural recruitment thresholds and expanded dynamic range, measured using electrically - evoked auditory brainstem responses). The basis for the efficiency of close-field electroporation arises from the compression of the electric field in proximity to the ganged cochlear implant electrodes. The regions close to the array with highest field strength corresponded closely to the distribution of bioreporter cells (adherent human embryonic kidney (HEK293)) expressing green fluorescent reporter protein (GFP) following gene electrotransfer. The optimization of the gene electrotransfer parameters using this cell-based model correlated closely with in vitro and in vivo cochlear gene delivery outcomes. The migration of the cochlear implant electrode array-based gene electrotransfer platform towards a clinical trial for neurotrophin-based enhancement of cochlear implants is supported by availability of a novel regulatory compliant mini-plasmid DNA backbone (pFAR4; plasmid Free of Antibiotic Resistance v.4) which could be used to package a ‘humanized’ neurotrophin expression cassette. A reporter cassette packaged into pFAR4 produced prominent GFP expression in the guinea pig basal turn perilymphatic scalae. More broadly, close-field gene electrotransfer may lend itself to a spectrum of potential DNA therapeutics applications benefitting from titratable, localised, delivery of naked DNA, for gene augmentation, targeted gene regulation, or gene substitution strategies

SIRNA-Directed In Vivo Silencing of Androgen Receptor Inhibits the Growth of Castration-Resistant Prostate Carcinomas

BACKGROUND: Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools

Cyclopentenyl cytosine increases gemcitabine radiosensitisation in human pancreatic cancer cells

The deoxycytidine analogue 2′,2′-difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a potent radiosensitiser, but has limited efficacy in combination with radiotherapy in patients with pancreatic cancer due to acute toxicity. We investigated whether cyclopentenyl cytosine (CPEC), targetting the ‘de novo' biosynthesis of cytidine triphosphate (CTP), could increase dFdC cytotoxicity alone or in combination with irradiation in a panel of human pancreatic cancer cells (Panc-1, Miapaca-2, BxPC-3). To investigate the role of deoxycytidine kinase (dCK), the rate-limiting enzyme in the activation of dFdC, human lung cancer cells without (dFdC-resistant SWg) and with an intact dCK gene (dFdC-sensitive SWp) were included. We found that CPEC (100–1000 nmol l−1) specifically reduced CTP levels in a dose-dependent manner that lasted up to 72 h in all cell lines. Preincubation with CPEC resulted in a dose-dependent increase in dFdC incorporated into the DNA only in dFdC-sensitive cells. Consequently, CPEC increased the effectiveness of dFdC (300 nmol l−1 for 4 h) only in dFdC-sensitive cells, which was accompanied by an increase in apoptosis. We also found that CPEC enhanced the radiosensitivity of cells treated with dFdC (30–300 nmol l−1 for 4 h). These results indicate that CPEC enhances the cytotoxicity of dFdC alone and in combination with irradiation in several human tumour cell lines with an intact dCK gene

Cellular processes of v-Src transformation revealed by gene profiling of primary cells - Implications for human cancer

<p>Abstract</p> <p>Background</p> <p>Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. In this study, we took advantage of several strains of the Rous sarcoma virus (RSV) to characterize the patterns of v-Src-dependent gene expression in two different primary cell types, namely chicken embryo fibroblasts (CEF) and chicken neuroretinal (CNR) cells. We identified a common set of v-Src regulated genes and assessed if their expression is associated with disease-free survival using several independent human tumor data sets.</p> <p>Methods</p> <p>CEF and CNR cells were infected with transforming, non-transforming, and temperature sensitive mutants of RSV to identify the patterns of gene expression in response to v-Src-transformation. Microarray analysis was used to measure changes in gene expression and to define a common set of v-Src regulated genes (CSR genes) in CEF and CNR cells. A clustering enrichment regime using the CSR genes and two independent breast tumor data-sets was used to identify a 42-gene aggressive tumor gene signature. The aggressive gene signature was tested for its prognostic value by conducting survival analyses on six additional tumor data sets.</p> <p>Results</p> <p>The analysis of CEF and CNR cells revealed that cell transformation by v-Src alters the expression of 6% of the protein coding genes of the genome. A common set of 175 v-Src regulated genes (CSR genes) was regulated in both CEF and CNR cells. Within the CSR gene set, a group of 42 v-Src inducible genes was associated with reduced disease- and metastasis-free survival in several independent patient cohorts with breast or lung cancer. Gene classes represented within this group include DNA replication, cell cycle, the DNA damage and stress responses, and blood vessel morphogenesis.</p> <p>Conclusion</p> <p>By studying the v-Src-dependent changes in gene expression in two types of primary cells, we identified a set of 42 inducible genes associated with poor prognosis in breast and lung cancer. The identification of these genes provides a set of biomarkers of aggressive tumor behavior and a framework for the study of cancer cells characterized by elevated Src kinase activity.</p

Giving Voice to Working Mothers: A Consumer Informed Study to Program Design for Working Mothers

Working parents experience considerable stress as they strive to cope with competing demands from work and family. However, workforce participation has shown to safeguard their personal wellbeing. It is therefore important that parents are adequately supported though appropriate and acceptable interventions in order to help them achieve quality of life, without needing to sacrifice one life domain for the other. This study adopted a consumer-focused perspective to program design to identify the relative fit between parental needs an existing workplace intervention. Focus groups were conducted with fifteen working mothers aged 30-44 years (M = 38.67). A thematic analysis revealed eight themes: (a) the impact of guilt, (b) crossover effects of work on family, (c) the availability of support, (d) being a quality parent, (e) getting the balance right, (f) impact on couple relationship, (g) having a career counts, and; (h) the need for low intensity programs. The extracted themes provided a good fit with existing workplace interventions that address stress and parenting. However, the results also indicated that working mothers need further assistance with strategies to manage guilt and the need to build on career strengths. Working parents also indicated a desire for briefer interventions. The implications for program design, including possible modifications, to current workplace interventions are discussed