Development of a cell-based treatment for long-term neurotrophin expression and spiral ganglion neuron survival
Introduction
Sensorineural hearing loss (SNHL), the most common form of deafness, is typically caused by the loss of cochlear hair cells. The only therapeutic treatment for patients with severe-profound SNHL is a cochlear implant – a neural prosthesis that electrically stimulates the residual spiral ganglion neuron (SGN) population to provide the rate and pitch cues necessary for speech perception. In the normal cochlea, the hair cells and supporting cells of the organ of Corti support the survival of SGNs through endogenous neurotrophin secretion (Fritzsch et al., 2004, Stankovic et al., 2004, Green et al., 2012, Zilberstein et al., 2012), and therefore damage to the organ of Corti and loss of this neurotrophin support, as occurs in SNHL, leads to the loss of SGNs. Since SGNs are the target cells for the cochlear implant, the loss of a significant population of SGNs may compromise the function of the device (Pfingst and Sutton, 1983, Shepherd and Javel, 1997, Hardie and Shepherd, 1999). Furthermore, future developments in device and software design may also benefit from an enhanced SGN population (Wise and Gillespie, 2012).
Exogenous delivery of neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) can support SGN survival in models of deafness (Ernfors et al., 1996, Miller et al., 1997, Gillespie et al., 2003, Gillespie et al., 2004, Yamagata et al., 2004, Richardson et al., 2005, Shepherd et al., 2005). However, the cessation of exogenous neurotrophin treatment can result in a loss of these survival effects (Gillespie et al., 2003, Shepherd et al., 2005). While others have reported continued auditory neuron survival for 2 weeks post-treatment (Agterberg et al., 2009), long-term outcomes remain unknown. Chronic electrical stimulation via a cochlear implant extends neurotrophin-based survival effects past the end of neurotrophin treatment; however, to maximize SGN rescue, long-term neurotrophin delivery is desirable (Shepherd et al., 2005, Shepherd et al., 2008).
Current methods of neurotrophin delivery into the cochlea, such as osmotic pumps, are not considered suitable for clinical application (Pettingill et al., 2007). Alternative pump-based delivery systems must be re-filled at regular intervals, necessitating multiple surgical procedures. This poses a small but significant risk of infection, which could result in labyrinthitis and meningitis (Wei et al., 2008). Furthermore, neurotrophins have a short half-life (Lindholm et al., 1988, Matsuoka et al., 1991, Poduslo and Curran, 1996, Kishino et al., 2001), meaning that the use of long-term pump delivery systems with high volume capacities may be complicated by the unknown bioactivity of neurotrophins maintained at body temperature for long periods.
Cell-based therapies, in which cells secreting a therapeutic substance are implanted into a patient, are an alternative mechanism for continuous delivery of neurotrophins into the cochlea (for review see Zanin et al., 2012). Cell-based therapies may utilize cells which naturally secrete therapeutic agents (Wise et al., 2011), or may be combined with gene transfer techniques to genetically modify cells to secrete the desired therapeutic agent(s) (Pettingill et al., 2008, Pettingill et al., 2011). Cell-based therapies provide an avenue for delivering neurotrophins at physiological levels and in a consistent manner, and also overcome issues of infection (Shepherd, 2011) and longevity of survival effects (Gillespie et al., 2003, Shepherd et al., 2005) associated with other experimental delivery methods. In addition, cell-based therapies have the potential for long-term neurotrophin expression (Winn et al., 1996). For these reasons, cell-based therapies are considered clinically viable, and have already been implemented for therapeutic drug delivery in clinical trials for various neurodegenerative conditions (for review see Zanin et al., 2012).
Previously, we successfully genetically modified Schwann cells using lipofection to express BDNF or NT3 and demonstrated that these cells could support SGN survival in vitro (Pettingill et al., 2008). Furthermore, we reported that the implantation of encapsulated, BDNF-secreting Schwann cells into the deaf guinea pig cochlea successfully supported SGN survival over 2- and 4-week implantation periods (Pettingill et al., 2011). While promising, longer term studies, using cells with a greater duration of neurotrophin secretion, are required in order to best assess the potential of this therapy for ongoing SGN survival (Pettingill et al., 2011).
There are numerous experimental parameters that may play an important role in achieving longer term neurotrophin expression from a cell-based treatment. In the current study, we investigated different (i) cell types, (ii) gene transfer techniques and (iii) neurotrophins, with the aim of developing a population of cells that reliably secreted neurotrophin for periods of time significantly greater than 4 weeks.
Section snippets
Experimental procedures
We utilized an array of test conditions in order to develop cells with long-term neurotrophin expression. Specifically, Schwann cells and fibroblasts were genetically modified using lipofection or nucleofection to express BDNF or NT3. Schwann cells were also genetically modified using lentiviral vectors to express these neurotrophins. The resultant neurotrophin-expressing cells were compared in terms of transfection efficiency, and duration and quantity of neurotrophin expression. The
Results
In this study we compared various approaches for cell transfection with the aim of developing a protocol that would generate cells with long-term neurotrophin secretion at levels sufficient to support SGN survival in vitro. Specifically, we genetically modified Schwann cells and fibroblasts to express EGFP, BDNF or NT3 using lipofection and nucleofection, and also genetically modified Schwann cells to express these genes using lentiviral vectors. We assessed the outcomes by quantifying the
Discussion
This study was undertaken to produce a population of primary cells that secrete neurotrophin for extended periods of time at concentrations that support SGN survival. We genetically modified Schwann cells and fibroblasts using lipofection and nucleofection, and also genetically modified Schwann cells using lentiviral vectors, to express two genes known to be important in the auditory system – BDNF and NT3. From the parameters tested we demonstrated that nucleofected BDNF-expressing fibroblasts
Conclusions
We utilized different methods of gene transfer to genetically modify Schwann cells or fibroblasts to secrete BDNF or NT3. We established that fibroblasts that were nucleofected to express BDNF provided the greatest duration of neurotrophin secretion of at least 30 weeks, and that these BDNF-expressing fibroblasts were effective in supporting SGN survival in an in vitro model of deafness, which indicates that therapeutic levels of neurotrophins can be delivered using cell-based methods. Since a
Author contributions
MPZ, RKS and LNG designed the experiments; MH and ARH produced the lentiviral Schwann cells used for the experiments; MPZ and LNG performed the experiments and wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgments
We thank Rebecca Argent for technical assistance, and Professor Johnson Mak for access to the AMAXA Nucleofector II. This work was supported by the National Health and Medical Research Council of Australia (APP526901) and the Garnett Passe and Rodney Williams Memorial Foundation. The Bionics Institute acknowledges the support it receives from the Victorian Government through its Operational Infrastructure Support Program. The funding bodies had no role in study design, data collection and
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Bridging the electrode–neuron gap: finite element modeling of in vitro neurotrophin gradients to optimize neuroelectronic interfaces in the inner ear
2022, Acta BiomaterialiaCitation Excerpt :Over the course of the past 20–30 years, it has been established that BDNF mediates survival and differentiation activities of SGNs by binding and activating tyrosine kinase receptor kinase B (TrkB), a member of the larger family of Trk receptors [20]. Numerous studies have reported that BDNF can palliate SGN degeneration in ototoxically deafened animals, a widely accepted model for retrograde trans-synaptic SGN degeneration secondary to hair cell destruction [13,14,57,58]. Additionally, it has been confirmed that there is a positive correlation between SGN counts and CI performance [59].
Recent advances in the implant-based drug delivery in otorhinolaryngology
2020, Acta BiomaterialiaCitation Excerpt :Like NIH3T3 cells, primary fibroblasts have also been successfully explored. One noteworthy study focused on the optimization of important parameters of a cell-mediated neurotrophin-delivering implant, i.e., cell type, gene transfer method, and neurotrophin type [24]. Their study concluded that nucleofecting fibroblasts to express BDNF yields the most sustained neurotrophin expression.
Scaffolds for auditory nerve regeneration
2019, Handbook of Tissue Engineering Scaffolds: Volume TwoCell-based neurotrophin treatment supports long-term auditory neuron survival in the deaf guinea pig
2015, Journal of Controlled ReleaseCitation Excerpt :Specifically, we combined cell- and gene therapies with alginate encapsulation technology to produce encapsulated BDNF-expressing fibroblasts, and assessed the survival-promoting effects on ANs in the deaf guinea pig following implantation for periods of up to six months, and with and without chronic electrical stimulation. Fibroblasts that were nucleofected to express BDNF were used for this study based upon findings from in vitro experiments [27], which demonstrated that these parameters were most efficacious in terms of gene transfer, and produced cells which had the greatest duration of neurotrophin expression. Specifically, fibroblasts were isolated from rat sciatic nerve explants using a method similar to that used to isolate Schwann cells [21,28].
Nucleic acid direct delivery to fibroblasts: a review of nucleofection and applications
2022, Journal of Biological Engineering
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Current address: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis, TN, USA.
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Current address: Laboratory for Transplantation and Regenerative Medicine, Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sweden.