- Sai Charan
Research: Inner Auditory Hair Cell Regeneration
Author(s): Adithya Acharya and Siddharth Acharya
For correspondence related to this article, please e-mail abcdef128495@gmail.com.
Abstract:
This essay gives a semi-detailed report of using Viral Vectors to regenerate inner ear hair cells to reverse the effects of hearing loss. The viral vector which will be discussed is AAV or Adeno – Associated virus, which has been proven to regenerate hair cells in mice.
The first quarter of this essay will discuss the need for an improvement in inner hair cell regeneration technology and current technology used for regaining audibility for the deaf (a good example would be of cochlear implants), and the advantages and disadvantages of the technology.
The next 3 quarters will discuss AAV and its benefits, along with vital experiments. It is followed with ATOH1, what it is, its benefits; then immunosuppressants, and finally, the financial burdens of it all.
Acronyms Used in this Document:
IHCR: Inner Hair Cell Regeneration (or) Inner Hair Cell Regeneration Therapy [Although Inner Hair Cells refer to all inner – body hair cells, in this essay we use this acronym for describing Inner Ear Hair Cells only]
AAV: Adeno – Associated Virus
IEGT: Inner Ear Gene Therapy
IHC: Inner Hair Cell
OHC: Outer hair cell
Glossary:
Ototoxic: Drugs that are harmful to the ear; heavy administration usually results in permanent hearing loss
Vestibular Functions/ Vestibular: Related to the inner ear / Sense of Balance
Basilar Papilla: Auditory systems of lower vertebrae like amphibians, lizards, and birds
Vibrotactile Device: Devices that give the feeling/perception of vibration through touch [0]
Viral Vectors: Means by which scientists deliver genetic material into cells
Immune Privileged Organs: These are sites in the human body that do not interrupt the immune system when a foreign agent is placed in it.
Immunosuppressants: Drugs that can disable the bodies immune response for a certain region or area of the body, in turn preventing inflammation and other immune response symptoms
Preface:
This document must be taken as a budding research paper. Research can be taken upon the topics discussed without the permission of the author. Nevertheless, any damage to property or living beings will not be entertained through this document, and the author bears no responsibility to any such.
Copyright:
No part of this document shall be reproduced, republished, or forwarded through any means without the electronic or written permission of the author.
How does this come as innovation in technology?
Nowhere, in absolutely no government or private document is there a mention of IHCR using a mix of AAV, ATOH1, and immunosuppressants. All parts of this essay have been delicately written to keep this entire essay factually correct, with a tint of innovation.
The main idea for this topic came from our own experiences and through the eyes of the patients. The world is obsessed with improving its overall technology, using high-tech robotics and mechanics to create marvels that would have been deemed as hoaxes just a few decades ago.
Yet, more than 500 million people in this world have some kind of a hearing defect (about 466 million are deaf, with 34 million of them being young children), and among them, very few have the financial stability and the opportunity to be able to get a cochlear implant, a device which enables those with cochlear damage to hearing properly.
The cochlear implant is not only a financial burden, but it also requires consistent maintenance and repairment (more about cochlear implants in the further sections).
Inner Hair Cell Regeneration (IHCR) serves as a definite solution to this problem, and this essay serves to give a semi-detailed uncensored tale of IHCR, and why it is the definite solution for hearing loss in the future.
Intro to Inner Hair Cell Regeneration:
In short terms, Inner Hair Cell Regeneration is the process of the regrowth of auditory hair cells which can supposedly reverse the effects of deafness.
These hair cells are also vital in balance. The inner ear sensory organs comprise of the ‘cochlea', ‘utricle', ‘saccule', and the ‘crista ampullar' of the three semicircular canals.
History and Necessity of Inner Hair Cell Regeneration:
Currently, hearing aids are the driving mechanism to remove the effects of hearing loss. Note that hearing aids only amplify the sound coming from the sources. Hearing Aids prove useless to reverse hearing loss when the complete cochlea is damaged. Hearing Aids also fail to detect certain frequencies, leading to disturbances in speech recognition.
To enable hearing for hearing – loss patients with damage in their cochlea, Cochlear – Implants are necessary.
Cochlear implants are mainly used for children with hearing loss conditions from an early age due to birth conditions, but it is also required in adults.
The basic mechanism of Cochlear Implants is by transmitting obtained sound waves into pulses by an external processor which must be worn by the patient. These transmitted pulses are received by an internal processor which transmits them to the brain in the form of electrical signals. Since there is an internal mechanism, surgery is necessary for this procedure.
Cochlear Implants prove highly effective with a dejection rate of 0.2 % and a need for re-installment of 0.5 %. However, stating cochlear implants as the best alternative for hearing loss would not be right.
Cochlear Implants are delicate and expensive equipment, and since it requires an external device, it also needs proper care and maintenance.
The external Cochlear implant consists of a microphone, sound processor, and a transmitter. To keep this connection with the internal system, a magnet is used. This could prove to be dangerous in certain circumstances, as exposure to strong magnetic fields could adversely impact the internal mechanism, at the same time posing a risk to the user.
The external processor also requires constant charging after use, so multiple external processors will be necessary for those who are continuously interacting with other peers (school–going children, office goers, workers, etc.). The constant repair for ‘wear and tear' could be exorbitant to a few.
We present Inner Hair Cell Regeneration Therapy as a decent alternative to eliminate the above disadvantages.
Feasibility of IHCR:
As we have stated above, normal regeneration of inner hair cells has not been observed. However, IHCR has been detected in lower vertebrae (such as amphibians, birds, reptiles, and fish). Experimental results have proven that hair cells regenerate in birds under normal conditions.
IHCR Experiments in Lower Vertebrae:
(Exp - 1) IHCR in birds was first referred to by Cotanche, who observed regeneration after noise-induced hearing loss. He perceived that damaged cells were extruded from the epithelium and germinal- basal cells replaced the damaged ones. An important point to be noted is that this process peremptorily, with the microvilli reappearing on the apical surface of the epithelial tissue 48 hours after the noise-induced deafness. Hair Cell Regeneration was completed after 10 days of the trauma.
(Exp - 2) The auditory sensory organs of amphibians, lizards, and birds (termed as Basilar papilla) consist of epithelial tissue in place of mammalian hair cells. Gentamicin intoxication is a toxic level that causes nerve, kidney, or renal damage along with permanent hearing loss in humans. The regeneration of the basilar papilla of birds after gentamicin intoxication was reported by Cruz and his colleagues, who reported complete recovery of the cells 3 weeks after the ototoxicity was caused.
Other experiments in lower vertebrae include regeneration of hair cells in adult parrots reported by Jorgensen and Mathieson, regeneration in chinchilla reported by López among others, but our point has been clearly stated.
IHC Regeneration in Humans and Experiments related with it:
In 1977, Moffat and Ramsden were the first to suggest regeneration in humans. Their report is on a 37 – year - old man with certain complications due to bilateral chronic kidney failure. Postoperative is a severe condition after surgery from a kidney transplant. The man developed an infection and was administered 240 mg of gentamicin (an ototoxic) over 10 hours. After a day of administration, the patient developed bilateral deafness. The first auditory test confirmed complete deafness in the left ear and severe neurosensory hearing loss in the right ear. Initially, his hearing showed a max threshold of 100 dB and very poor cochlear microphonics. However, three weeks later his hearing improved with sensations from 125 to 500 Hz; after 8 months the threshold of his audible frequency was around 70 dB.
In 1980, a scientist reported a series of 138 patients who were each administered tobramycin and gentamicin, both ototoxic. 55% of these patients recovered their audible sensations between a week to 6 months and 53% recovered their vestibular function between 10 days and 9 months after ototoxic administration.
Two Possible Techniques which could be implemented:
1) Use of an internal mechanism: Currently, hearing aids and cochlear implants serve as well–known ‘machinery' which are used to weaken the effects of hearing loss. Middle Ear Implants, Bone Anchored Hearing Aids, Auditory Brain Stem Implants all serve as alternatives to cochlear implants, although they are not much improved. Implants may seem like a solution to hearing loss, but there exists a better alternative to it.
2) Use of external viral vectors:
Disadvantages of Implants:
As an external processor is involved, the frequent repair is necessary.
To attach the internal and external mechanisms, a magnet is usually preferred. This could prove dangerous when exposed to high magnetic fields, like in MRIs.
To implant the internal mechanism, an operation is involved.
Advantages of Implants:
Since this method has been in use for a long time, its procedure is well known and approved.
Its failure rate stands at about 4.8 %, far less than other surgeries.
Its cost is moderately average and affordable to most.
The probable best alternative to this would be by using Viral Vectors [Definitions given above].
The viral vector which could be used is AAV or Adeno-Associated Virus. It has been successfully used in mice whose hearing loss is hereditary. AAV2.7m8 is a synthetic AAV that infects both inner hair cells and outer hair cells with high efficiency. It is important and necessary to note that the AAV virus only delivers the gene, but the main regeneration process is performed by the gene. In short terms, AAV2.7m8 is used as a delivery agent and the gene is the delivered agent which will operate the regeneration.
The mammalian cochlea consists of two types of hair cells, named inner hair cells (IHC) and outer hair cells (OHC), both of which are necessary to detect and process auditory information. These hair cells are surrounded by supporting cells, a group of cells that are vital for stability in the cochlea. Since the mature mammalian hair cells are not capable of regeneration, regeneration of these cells using viral vectors (termed as Inner ear gene therapy) proves promising.
Inner ear gene therapy (IEGT) can potentially prevent and reverse hair cell damage in mammals. Most studies studying IEGT using viral vectors refer to AAV for gene delivery. AAV is a single-stranded DNA parvovirus belonging to the genus Dependoparvovirus. It is used in therapies because it is non-pathogenic in humans. AAV serotypes have been shown to infect IHCs much more effectively than OHC.
For AAVs to achieve complete hearing restoration, the efficiency must be improved. High levels of effort have been put to enhance the efficiency of AAVs, which led to the production of synthetic AAVs.
Through certain procedures, two synthetic AAVs which have been developed include AAV2.7m8 and AAV8BP2. AAV2.7m8 can infect the inner pillar cells and inner phalangeal cells with high efficiency. These results suggest that AAV2.7m8 is a useful vector for IEGT. The AAV2.7m8 vector contains a 10-amino acid peptide inserted in the AAV2 capsid protein sequence.
Details of AAV (or) Adeno – Associated Virus:
Experiments involved in the testing of AAV: The main and primary experiment involved in the testing of AAV for regeneration of audibility would be in mice.
To check the infection efficiency of synthetic AAVs in the mammalian inner ear, AAV2.7m8-GFP among other varieties of AAV were delivered into the inner ears of newborn mice through the vertical semicircular canal. Using the posterior semicircular canal, the viral vectors are delivered to infect cells in the cochlea along with the other vestibular organs.
One microliter (or 10-6 liters) of AAV was delivered into each mouse (In the further sections of this essay where we discuss cost, we will find out that for even 100 microliters, the cost is exceptionally low). To find out the hair cell infection efficiency, light in the range from blue to ultraviolet was shone to detect the emission of bright green fluorescence emitted by the green fluorescent protein. Examination of the cochlea after 4 weeks of gene delivery revealed high levels of fluorescence in both the inner hair cells and outer hair cells.
The overall infection efficiency was about 78.9% to 89.76% for inner hair cells and 72 – 89.8% for outer hair cells.
Delivery Gene – ATOH1: As we have stated above, the main regeneration is undertaken by the gene whereas the gene is delivered using AAV2.7m8. The most probable gene which could be used is Atonal Homolog 1 (ATOH1).
ATOH1 is required for the formation of both neural and non-neural cell types, and it has been proven to regenerate inner ear hair cells.
Hence it would be a possible gene for IHCR, transported by AAV2.7m8.
Drawbacks of IHCR:
Side – Effects of using AAV and ATOH1: Like almost all things in this universe, the procedure of IHCR has a few drawbacks, in the form of side – effects.
The main side–effect itself is suggested as ‘incorrect' or ‘impossible' by previous old dogmas and rumors. It is believed that the ear is ‘immune – privileged, which is a fancy word for saying that any foreign substance does not interrupt or disrupt the immune system to cause immune responses.
This thought is undoubtedly wrong, or incorrect in some manner - When viruses or external bodies do enter the cochlea, an immune response like inflammation may occur.
The old dogma comes from the fact that neutrophils do not enter the cochlea. However, bone marrow–derived resident macrophages are always present in the spiral tissues of the walls of the cochlea.
Prevention of Immune Responses: To prevent this immune response, we could add a solution of diluted regulatory T -cells while delivering the viral vectors into the ear. This would prevent a minor to massive immune response without diminishing the quality of the IHCR vectors.
Regulatory T – Cells ‘regulate' immune responses in the immune body (immune responses can be caused by effector T – cells). In the human body, they are produced in the bone marrow. For our IHCR project, it will be essentially important to make sure that side – effects are kept at bay. To do so, the immune responses which could be caused by the macrophages can be rightly suppressed using Regulatory T – cells. It is highly important to note that the quantity of such T -cells must be carefully moderated. Excess of T - cells are proven to hinder the human body's inbuilt immunity against tumor cells.
It can be also noted that as science improves, better and better anti–immune response drugs can and have been produced, which could possibly be safer than regulatory T -cells. These types of ‘immune-response suppressing' drugs are coined as immunosuppressants.
Alternative Immunosuppressants (other than regulatory T – cells): Otic Corticosteroids, Azathioprine, Cyclophosphamide, Methotrexate.
Immunosuppressants are slightly expensive in nature, so they may add a bit to the financial burden.
Feasibility and Costs of IHCR:
In the present era, cochlear implants are acquired by the deaf through surgery, similarly, IHCR requires the accord from specialists in the relevant field. It is definitely illegitimate to supply the drugs of IHCR in the free market. Only affiliated pharmaceutical industries and hospitals have the authorization to own and prescribe. The costs discussed here are based on each individual component vital for IHCR. Since the effectiveness of AAV and the gene is exceptionally high, even 1 microliter (10-6 liters) would be adequate.
Conclusion:
To conclude, properly assembling IHCR centers and completely alternating mechanical devices with Inner Hair Cell Regeneration would be a huge leap in demolishing barriers faced by hard–of–hearing patients along with deaf patients. It would also be economically satisfying, both to the government and patients.
Perhaps the best part of IHCR is the fact that once inner ear hair cells do re-establish themselves, it requires minimal maintenance. Compared to regular repairing and mending of cochlear implant patients, it would be highly acceptable to have a procedure that fixes the problem through the body rather than keeping either an external or internal device.
For all we know, IHCR must come as a better alternative to other forms of hearing implants, and for the better.
Bibliography:
https://arizonahearing.com/scientists-discover-way-to-regenerate-inner-ear-hair-cells/
https://www.eurekalert.org/pub_releases/2019-12/meae-rie120419.php
https://www.bcig.org.uk/cochlear-implant-devices/implantable-devices/#SemiImplantable
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146031/#:~:tible
https://www.frontiersin.org/articles/10.3389/fphar.2014.00287/full
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