Cellular targeting for cochlear gene therapy

Abstract

Gene therapy has considerable potential for the treatment of disorders of the inner ear. Many forms of inherited hearing loss have now been linked to specific locations in the genome, and for many of these the genes and specific mutations involved have been identified. This information provides the basis for therapy based on genetic approaches. However, a major obstacle to gene therapy is the targeting of therapy to the cells and the times that are required. The inner ear is a very complex organ, involving dozens of cell types that must function in a coordinated manner to result in the formation of the ear, and in hearing. Mutations that result in hearing loss can affect virtually any of these cells. Moreover, the genes involved are active during particular times, some for only brief periods of time. In order to be effective, gene therapy must be delivered to the appropriate cells, and at the appropriate times. In many cases, it must also be restricted to these cells and times. This requires methods with which to target gene therapy in space and time. Cell-specific gene promoters offer the opportunity to direct gene therapy to a desired cell type. Moreover, conditional promoters allow gene expression to be turned off and on at desired times. Theoretically, these technologies offer a mechanism by which to deliver gene therapy to any cell, at any given time. This chapter will examine the potential for such targeting to deliver gene therapy to the inner ear in a precisely controlled manner.

Figure 1

Schematic representation of gene regulation. The core promoter of a gene lies immediately upstream of the transcription initiation site at which mRNA production by RNA polymerase II (Pol II) begins. The promoter serves as a binding site for the transcription complex, which consists of Poll II, various transcription factors (TFs), and for promoters containing a TATA box, the TATA-binding protein (TBP). At other locations in the gene, enhancers serve as binding sites for additional TFs and can influence the rate of transcription of the gene. Binding of different combinations of TFs can direct expression to different cell types or times.

Figure 2

Schematic representation of the means by which enhancers are thought to influence the transcriptional complex (TC). Transcription factors (TFs) that are bound to their DNA recognition DNA motifs in an enhancer are brought into contact with the TC by the folding of DNA. Either the TFs or a transcription-associated protein (TAP) interact with the TC via a transactivation domain. Many promoters are inactive without the influence of enhancers.

Figure 3

Expression of green fluorescent protein (GFP) driven by regulatory elements from the atoh1 gene, in the inner ear of transgenic mice. a Entire membranous labyrinth at E13.5. The sensory organs of the vestibular system, including the utricle (utr), saccule (sac), and crista, which contain differentiating hair cells (HCs), express GFP. The more immature cochlea (coch), in which HCs have yet to develop, does not. b Dissected cochlea at E14.5. HCs have now developed through 1.5 turns. The arrow indicates the basal region where HC differentiation initiates, arrowhead indicates the leading edge of HC differentiation as shown by the extent of GFP-expressing HCs. Scale bars equal 100 µm. c Confocal image of E176.5 organ of Corti [27].

Figure 4

Expression of green fluorescent protein (GFP) driven by the human elongation factor 1α (ef1α) promoter in the organ of Corti of the mouse. Plasmid DNA was electroporated into the E11.5 mouse otocyst. The inner ear was fixed and processed for histochemistry at E17.5 [59].

Figure 5

Expression of yellow fluorescent protein, driven by an altered thy1 promoter, in the neurons of the neonatal spiral ganglion of a transgenic mouse. a Neuronal somata in the spiral ganglion (SG) are intensely labeled, as are afferent dendrites (AD) and a dense plexus of nerve terminals underneath the inner hair cells (HCs) in the organ of Corti (oC). b Higher magnification image of SG neurons and their dendrites, with branching terminations under the inner HCs characteristic of this developmental stage.