Gene Therapy

Background

In mammals, the limited number of hair cells ( 15,000 in humans) develop during a narrow window of time in embryonic life. In contrast, hair cells of most nonmammalian vertebrates (fish, amphibians, and birds) either continue to form throughout life or regenerate after damage or death by proliferation and differentiation of the supporting cells. This finding that hair cells can regenerate in nonmammalian vertebrates has triggered many studies on gene therapy for treating or curing deafness.

Some researchers believe that the small size of the inner ear and its location and structural complexity limit the access and therefore the potential treatments for sensorineural hearing loss (SNHL). But the more common opinion is that the inner ear is an attractive target for gene therapy for these very same reasons [1-2]. Some of the advantages of the inner ear as a site for gene therapy include the following:

• damage to the organ of Corti, a small and localized organ, is responsible for most cases of HL;

• the potential spread of gene transfer viruses (i.e., vectors) to adjacent tissues is limited due to the relative isolation of the inner ear, provided by the surrounding otic capsule;

• the presence of the perilymphatic and endolymphatic liquid spaces may allow functionally important cells to be accessed by a transfection reagent;

• physiological measurement tools are available to evaluate the function of many of the cells targeted by gene therapy. These tools include cochlear microphonics (CMs) and otoacoustic emissions (OAEs) to assess OHC function; compound action potentials (CAPs) for IHC function; speech discrimination tests for detecting auditory neuropathy (SGN function); detection of endocochlear potentials (EPs) to determine endolymphatic ion balances; and the diagnostic tool ABR, which records changes in all parts of the auditory pathway and enables differentiation of cochlear from retrocochlear hearing disorders.

Thus, introducing gene therapy vector into the tiny volume of cochlea could be a possible way to restore the damaged cells in the cochlear or in the associated SGNs.

Method -- introducing gene therapy vector through the round window or the cochlear bone

Figure 1 Routes of delivery for cochlear gene therapy

The delivery of a therapeutic agent to the cochlea can be performed by direct application of the agent through an intact round window membrane (RWM) with (A) a gelation sponge, (b) direct injection through  the RWM, and  (C) infusion with an osmotic minipupm through a cochleostomy.  

Figure 1 shows the possible ways to introduce gene therapy vector into the cochlea. However, the round window (RW) should be exposed in order to do any gene therapy injection. The RW is accessible via the tympanic bulla of the rodent middle ear. The temporal bulla is opened through a postauricular incision to expose the round window. The round window membrane (RWM) is seldom damaged by this surgery [2].

Several ways can be used to introduce gene therapy gene:

• Direct microinjection: one way of intracochlear delivery of the vector carrying the gene to be expressed (vector-transgene complex) is by direct microinjection through the RWM. However, direct microinjection can easily rupture the RWM, causing a perilymphatic fistula.

• Mediated by Gelfoam: a less invasive technique for the delivery of therapeutic agents to the cochlea uses a small piece of Gelfoam that is placed on the RWM and soaked with the vector to be absorbed by simple diffusion. The Gelfoam technique proved to be an easy and effective method of delivering transgenes through an intact RWM [3-4]. The disadvantages of the Gelfoam technique are that gene expression following these experiments is mostly restricted to the basal turns of the cochlea, and the Gelfoam technique is vector-dependent. Transgene expression was obtained with Gelfoam absorbed with liposomes and adenovirus, but not with adeno-associated virus (AAV) [3-4].

• Injection of vectors to the scala tympani through the cochlear bone: this is achieved through a cochleostomy, a small hole in the cochlear bone. Substances can be directly injected or delivered over several weeks to the cochlea via osmotic minipumps that allow for long-term, steady, intracochlear delivery. Both delivery techniques of the vector-transgene complex are usually associated with localized surgical trauma and limited inflammation. Rare complications can result in loose or detached connective tissue, fibrosis and inflammation, ossification, atrophy of the stria vascularis, and cellular degeneration of the neuroepithelium [3-6].

• Cochleostomy: access to the scala media is also possible by a cochleostomy through the stria vascularis. However, delivery of adenovectors into the scala media seems to damage hair cells but improves transgene expression in other cell types in the organ of Corti [1].

Candidate genes involved in cell cycle regulation and differentiation

There are several studies demonstrate an ability to overproduce hair cells in the mammalian cochlea following elimination of specific cell cycle inhibitors. They are summarized in Table 2 below. However, the overproduction of hair cells appears transient and is followed by premature death of these cells as well as death of the animal at birth.

Table 2 Summary of positive and negative regulating genes appeared in published studies

Regulator	Function	Gene candidates
Positive regulation	Precursor cell differentiation regulation genes: cochlear hair cells and supporting cells arise from common precursors	Pou4f3, Pax2, Nkx5.1, Atoh1
	Maturation and survival of hair cells	Mutation of the Pou4f3 gene
	Differentiation of hair cells from its multipotent progenitors [7]	Atoh1
	Basic helix-loop-helix encoding [8]; regeneration of new hair cells [9]; cellular and functional repair in the organ of Corti [10]	Atoh1
Negative regulation	Cell cycle inhibitors regulate mitosis by stopping cell proliferation, leading to a mature, nondividing sensory epithelium [11]	p27/Kip1, pRb
	Initiation of DNA synthesis in hair cells but it is folowed by apoptosis of these cells resulting in HL[12]	p19/ink4d

Vectors for inner ear delivery

Viral and nonviral gene transfer vectors are used for delivery of gene therapy. The viral vectors capitalize on the natural infectivity of these viruses to introduce and express exogenous genes inserted within the viral genome. In vivo intracochlear gene transfer in various studies showed differences in efficacy, utility, and safety between the vectors studied. To date, there is no single vector available that is flexible enough to deliver all the diverse gene therapy substances to critical areas in the auditory system. The unique characteristics of each vector determine its efficacy and degree of safety, as well as the transgene expression. The characteristics of the different vectors are summarized in Table 1. Most studies are currently being performed with adenoviruses, although there are still ongoing experiments with AAV and Herpes simplex virus (HSV) vectors because of their potential ability to achieve long-term expression [13].

Table 1 Summary of the characteristics of different vectors [14]

Vector	Genome	Description of vector	Introduction to cell	Results of gene transfer to inner ear
retrovirus	RNA	Contains three conserved genes required for their normal life cycle: gag, pol, enu. The RNA genome is surrounded by a nucleocapsid core and a glycoprotein envelope	Attaches to host cells via the envelope surface protein and enters the cell by receptormediated endocytosis. Some retroviruses are amphotropic (able to transduce cells across species) and they recognize specific cell surface receptors. In the cell, a dsDNA intermediate is synthesized from the RNA template and the nucleoprotein complex enters the cell nucleus.	As the neurosensory epithelia of the inner ear is postmitotic (not dividing), it is not suitable for retroviral vector gene transfer
Lentivirus	RNA	Based on HIV	Stable, long-term expression of a transgene. No toxicity.	Gene transfer to the cochlea was restricted to the periphery of the perilymphatic space
Adenovirus (AV)	dsDNA	A common human pathogen causing a benign respiratory infection. Relatively stable. Linear dsDNA of about 36 kb. The virion is a capsid composed of protein and DNA. Easy to produce.	Binds to a receptor on the target cell for transfection. High transduction efficiency. Relatively safe. Temporally transgene expression (up to a month). Provokes a strong immune response, might be toxic to the recipient cell	Accomplished gene transfer to the widest variety of inner ear cells, including auditory and vestibular hair cells.
Herpes simplex virus (HSV)	dsDNA	Two types: replicationdefective recombinant viruses and plasmidderived amplicons. Hard to produce. Cytopathic nature	Nonpathogenic to neural tissues. Can mediate transgene expression in neurons and other cell types. Low infection efficiency	Expression of the transgene was observed in the supporting cells of the organ of Corti, in the spiral ganglion neurons, and in the stria vascularis. Herpes virus vectors appear to target neurons most efficiently.
Adeno-associated virus (AAV)	ssDNA	has a broad host range	Requires coinfection with adenovirus or HSV for lytic growth. Nonpathogenic in both humans and animals	Was found suitable in mediating in vivo transgene expression within the mammalian cochlea. Reporter gene expression was detected in most cell types in the cochlea including the spiral ligament, spiral limbus, organ of Corti, and SGNs. No expression was detected in the stria vascularis. The reporter gene expression was present up to 24 weeks following introduction. Higher expression at the basilar turn of the cochlea.
Liposomes	RNA/DNA	Cationic lipid vesicles. Non-imunogenic. Easily prepared in large amounts. Can be combined with any size DNA, resulting in stable complexes held together by ionic interactions	Binding of the liposome-DNA complex to the plasma membrane results in transfection of many cell types. Minimal risk of insertional mutagenesis. Liposome vectors are less efficient in terms of gene transfer compared with other vectors and there is no control over tissue targeting other than the site of vector delivery.	Transgene expression in the guinea pig cochlea was persistent up to 14 days in the neurosensory epithelia and surrounding tissue without toxicity and inflammation in the target organ

Growth Factors

Growth factors normally involved in embryonic development, including fibroblast growth factor, insulin growth factor, brain-derived neurotrophic factor, epidermal growth factor, and transforming growth factor-a, have been used either individually or in combination to determine whether they can induce regeneration by stimulating proliferation in supporting cells [15]. Only limited success has been achieved using this approach in the mature mammalian auditory sensory epithelium. New hair cells were generated in situ by mitotic cell division in adult mammals, but only a small number of the supporting cells retained an intrinsic capability to proliferate in the mature mammalian inner ear.

Reference

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[5] Ernfors P, Duan ML, ElShamy WM, Canlon B. "Protection of auditory neurons from aminoglycoside toxicity by neurotrophin-3". Nat Med 1996;2:463–467

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[7] Fekete DM. "Making sense of making hair cells". Trends Neurosci 2000;23:386

[8] Bermingham NA, Hassan BA, Price SD, et al. "Math1: an essential gene for the generation of inner ear hair cells". Science 1999;284:1837–1841

[9] Izumikawa M, Minoda R, Kawamoto K, et al. "Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nat Med 2005;11:271–276
[10]  Chen P, Segil N. "p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti". Development 1999;126:1581–1590

[11] Mantela J, Jiang Z, Ylikoski J, Fritzsch B, Zacksenhaus E, Pirvola U. "The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear". Development 2005;132:2377–2388

[12] Parker MA, Cotanche DA. "The potential use of stem cells for cochlear repair. Audiol Neurootol" 2004;9:72–80

[13] Maiorana CR, Staecker H. "Advances in inner ear gene therapy: exploring cochlear protection and regeneration". Curr Opin Otolaryngol Head Neck Surg 2005;13:308–312

[14] Zippora Brownstein, KarenB. Avraham. "Future Trends and Potential for Treatment of Sensorineural Hearing Loss". Seminar in hearing in  hearing. Vol. 27, no. 3, 2006

[15] Bermingham-McDonogh O, Rubel EW. "Hair cell regeneration: winging our way towards a sound future". Curr Opin Neurobiol 2003;13:119–126