AudNOWARC 2017a.ppt - Audiology ARC handout.pdf3/13/2017 1 BRENDA M. RYALS, PH.D. APRIL 5, 2017 ARC Disclosure I have relevant financial relationship(s) with the products or services

3/13/2017

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BRENDA M. RYALS, PH.D.APRIL 5, 2017 ARC

Disclosure

I have relevant financial relationship(s) with the products or services described, reviewed, evaluated, or compared in this presentation. James Madison University

Employed and receives salary.

NIDCD/NIH Funded research included in this presentation.

I have no relevant nonfinancial relationship(s) to disclose.

The discovery of hair cell regeneration in 1988 contradicted everything we knew about hair cell loss and permanent sensorineural

hearing loss.

It ain't what you don't know that gets you into trouble. It's what you know for sure that just ain't so.

Mark Twain

(-Once you lose a hair cell it is permanent;Temporary threshold shift isn’t permanently damaging, etc.)

Hair cell regeneration: The next step –translating this to the mammalian ear

B. Declan (2008) Nature 453:840‐842

Environmentalgraffiti.com

Reminder – what we learned from birds…Direct transdifferentiation

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G1G2

Mitotic regeneration

• Endogenous precursor cells (supporting cells)

– Mitosis (cell division)

(cartoons retrieved from: http://ronney19.bol.ucla.edu/BackgrndInfo.htm

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Reminder – what we learned from birds…

Direct transdifferentiation

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– Transdifferentiation (conversion)


Factors involved in regulating Cell Cycle

So in birds we have precursor cells (supporting cells) which respond to either to a release of inhibition (genetically controlled tumor suppressor –mitotic regeneration)

Factors involved in regulating Cell Cycle OR precursor cells that

respond to the stimulatory effects of other factors (morphogens; transcription factors) to dedifferentiate and become hair cells through conversion

• Morphogens are diffusible molecules that regulate cell fate determination during development.

• Transcription factors are proteins involved in the process of converting, or transcribing, DNA into RNA.

So here’s what we know about hair cell regeneration in birds:

•Precurser cells (supporting cells) are triggered to either re-enter the cell cycle or dedifferentiate and convert to hair cells when mature hair cells are damaged or destroyed


So here’s what we know about hair cell regeneration in birds:

Neural re-innervation of these newly formed cells essentially replicates embryonic developmental patterns with an initial abundance of afferent synapses followed by pruning. Efferent synapses remain in place and reform synapses.


Hair cell regeneration in birds essentially recapitulates development

Patrick J. Atkinson et al. Development 2015;142:1561-1571

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Regenerative capacities of

the adult mouse

cochlea, the chicken

basilar papilla and the

zebrafish lateral line

system.


© 2015. Published by The Company of Biologists Ltd

Proposed mechanisms of endogenous hair cell regeneration.



Takasaka and Smith, 1971Tanaka and Smith, 1978

Put a real mammal pic here

Are there precurser cells in the mammalian organ of Corti?

Establishment of In Vitro Modelsof Sensory Epithelium

•All cells in the mammalian sensory epithelium CAN be induced to become

hair cells. IF….– Immature – only up to post-natal 7-10 days

– Cells are released from local signals – genetic inhibition

– Presence of genetic signal for hair cell differentiation

White et al (2006) Nature; Doetzlhofer et al (2009) Dev. Cell

Strategies:1. Factors regulating cell cycle

(mitosis)2. Factors regulating cell fate

(transdifferentiation)

B. Declan (2008) Nature 453:840-842

What we learned from birds –translated to mammals

Endogenous precursor cells (supporting cells)

• BUT –Mitotic hair cell regeneration is prevented in mammals by:• Local factors = cell cell interactions• Tumor suppressor genes

• p27kip1• pRb1• p19ink4d (cartoons retrieved from:

http://ronney19.bol.ucla.edu/BackgrndInfo.htm

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The first tumor suppressing gene/protein associated with hair cell regeneration was p27 kip1

Mice deficient in the gene that regulates this protein developed too many hair cells (Segil et al 1999)

Retinoblastoma gene (pRb1) a tumor suppressing protein related to p27kip1 was associated with hair cell generation in embryonic mice (Sage et al 2005) but these hair cells died rapidly after production (Weber et al 2007).

Cell Cycle Control -

Figure 3. Ink4d is essential for actively maintaining the post-mitotic state of hair cells.a−f, Sections through representative P10 organ of Corti from wild-type (a and d), Ink4d-/- (b and e) and Kip1-/- (c and f) animals. a−c, Sections were double-labelled with antibodies against BrdU (green) and Myosin VIIa (red). d−f, Comparable sections were stained with an antibody against activated Caspase 3, a marker of apoptotic cells. Arrowheads indicate inner hair cells and brackets indicate outer hair cells. Arrows indicate a BrdU-positive hair cell (b) and an activated Caspase 3-positive hair cell (e) in Ink4d-/- animals, and BrdU-positive supporting cells in a Kip1-/-

animal (c). Scale bar represents 50 microns

p19Ink4d is essential for actively maintaining the post-mitotic state of hair cells (Chen et al 2003).

Hair cells in mice deficient in the tumor suppressor gene regulating this protein showed hair cell proliferation –BUT apoptosis rapidly followed

In the dish and in the embryo –Challenge for this work

What’s different about early development?

•Factors work to stimulate cell division and fate in early development but the cells don’t survive to maturity.

Not only do we need to find genes that inhibit cell division but we also need to understand how genes maintain hair cells in maturity

White et al 2012

Comparison of the modes and capacity of hair cell regeneration in the neonatal and adult mouse cochlea and

utricle.



There’s something different about babies

What we learned from birds – translated to mammals

Mitotic hair cell regeneration is prevented in mammals by genetic and local inhibition:

Challenges:•Find the right tumor suppressor gene•Find the right maintenance gene•How do we turn the inhibition back on?

•Tumor formation?•Maybe there is a “natural” local inhibition?


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What if we didn’t have to release inhibition? Could we convince cells to “change their

fate”?


• Transdifferentiation (conversion)• Morphogens


Factors involved in regulating cell differentiation or cell fate

Math1 (Bermingham et al 1999) more recently termed Atoh1

Notch Delta pathway (Hes1 and Hes5) (see for review Kelly 2002)

Genetic transcription factors available embryonically but “turned off” in mature animals Kelly, MW (2002) The Scientific

World Journal, 2;1079-1094

Atoh1 is the Hair Cell Gene

Without Atoh1 no hair cells are formed.

Other genetic transcription factors and morphogens regulate cell fate and work with Atoh1


Endogenous precursor cells (supporting cells)

• BUT – Hair cell regeneration through conversion is prevented in mammals by:• Morphogenetic signaling turned off after

embryogenesis• Transcription factors regulating early cell

division and differentiation down regulated in adulthood



• Conversion

• Challenges for signaling cell fate• How do we turn on morphogens?• How do we control transcription factors?• Does signaling order matter?• Which morphogens/transcription factors are

critical?cartoons retrieved from: http://ronney19.bol.ucla.edu/BackgrndInfo.htm

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All the cells in the mammalian cochlea are precurser cells under the right

circumstances

B. Declan (2008) Nature 453:840‐842Environmentalgraffiti.com

Released inhibition

Morphogenetic signals

•Strategies:1. Stem Cell Therapy – introduce

exogenous cells not inhibited from cell division

2. Gene Therapy – introduce morphogen or control transcription to stimulate an

endogenous cell to change fate3. Molecular Therapy - small molecule

targeting of developmental pathways for cell differentiation

B. Declan (2008) Nature 453:840‐842Environmentalgraffiti.com

https://youtu.be/M_VbUpYQDwA

Animation of Notch and Delta signaling

– Hypothesis for Hair cell generation after damage using developmental signals – Notch/Delta, Wnt etc.

Strategies:1. Factors regulating cell cycle (mitosis)

Stem Cell Therapy2. Factors regulating cell fate

(transdifferentiation)Gene Therapy

Molecular Therapy

B. Declan (2008) Nature 453:840-842

Stem Cells Definition: Stem cells are

characterized by their capacity to self-renew and their ability to differentiate asymmetrically to form cell types other than their own.

What are stem cells? How can they be used for medical benefit? - YouTube

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Stem Cell Therapy in the Cochlea

Can we find a stem cell niche in the inner ear? “stem cells can be isolated from vestibular sensory epithelia and from neonatal cochlea”

Ronaghi, Nasr and Heller 2012 Stem Cells 30:69-74

Differentiated cells can be obtained from a patient with a specific disease and reprogrammed to become iPS cells. The resulting iPS cells can then be in vitro differentiated to various specialized cell fates, such as neurons, cardiomyocytes or hepatocytes, and used to gain new insights into disease process or as cell‐based platform to develop new disease therapies.

Nobel Prize in Medecine and Phsyiology to Gurden and Yaminaka for iPS cells http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/advanced.html

Induced Pluripotent Stem Cells

Pre‐curser cells – Stem Cells – translation?

Exogenous ‐Endogenous

Oshima K, Shin K, Diensthuber M, Peng AW, Ricci AJ, Heller S. Cell. 2010 May 14;141(4):704‐16.

Hair cell-like cells (with stereocilia bundles and mechanosensory currents) can be derived from ES and from iPS cells (Induced Pluripotent Stem Cells – 2012 Nobel Prize in Physiology or Medicine to Gurdon and Yamanaka)

The Promise of iPS cells

•Available and no problem with immuno-reaction

•Abundant

•Can be made from humans with the disease/disorder

•Investigators have been successful in using iPS cells to form auditory neurons (Nishimura et al 2009) and hair cells (Oshima et al 2010)

Nishimura et al (NeuroReport 2009); Oshima et al (Cell 2010)

Stem Cell Therapy in the Cochlea

External application of human ES-cell-derived otic progenitors provides restoration of ABR in auditory neuropathy model. Chen et al 2012 Nature 490:278-282

As a general rule, stem cells transplanted into the cochlea tend to go to the modiolus –appear driven to a neural fate Martinez-Monedero and Edge 2007

(review); Corrales et al 2006

Results from several labs suggest mixed results showing in some but not all cases that transplanted neural stem cells can grow to make contact with hair cells in cochlear or vestibular hair cells and/or first order cochlear nucleus

Cartoons from the Stanford Initiative to Cure Hearing loss

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Using Cochlear Implants as a vehicle to induce neural growth/grow new neurons

Hair cell regeneration

apexbase

The spiral ganglion neurons must still be alive for the new hair cells to communicate to the brain.

Animation by Green 2005 with permission


apexbase

Moreover, the spiral ganglion neurons must be able to establish functional synaptic connections with the regenerated hair cells.

If most axons fail to connect, hearing will not be restored



apexbase

Disorganized connections will disrupt communication of frequency information




apexbase


Functional connections restore hearing


Challenges for the use of stem cells for hair cell

replacementAvailability*Delivery** Integration into site of

lesion – endolymph is toxic to HC

Driven to neural fate –likely more successful for neural regeneration

*iPS cells likely make this much less problematic.**McCall et al Drug Delivery for Treatment of Inner Ear Disease: Current State of Knowledge, Ear and Hearing. 31(2):156-165, April 2010.

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Gene Therapy

Gene therapy is an experimental treatment that involves introducing genetic material into a personʼs cells to fight disease

A gene can be delivered to a cell using a carrier known as a “vector.”The most common types of vectors used in gene therapy are viruses.

How Does Gene Therapy Work?

http://ghr.nlm.nih.gov/handbook/therapy/procedures

A new gene is injected into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.

Pre‐curser cells – Can we teach an old supporting cell a new trick? Gene Therapy/Molecular Therapy Exogenous ‐ Endogenous


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Doetzlhofer et al. Dev. Bio. 272 (2004)

What kind of genetic signaling would help to restore hair cells to a damaged cochlea?

Morphogens and Transcription factors – hair cells Inhibit Notch/Delta and Shh

signaling Enhance Wnt, FGF and

Atoh1 Morphogens and Transcription

factors – support cells Enhance Hes1, Hes5 and

FGFBreuskin et al 2008 Hearing Research –Review paper Genes and Regeneration

Gene Therapy in the Cochlea

Yehoash Raphael and colleagues (2003) were the first to report success with gene therapy for regenerating hair cells. They injected Atoh1 into damaged guinea pig cochleae and saw new hair cells develop in damaged regions. In their second experiment they confirmed functionality of hair cells with ABR (2005)

Kawamoto et al., J. Neurosci 23:4395, 2003Izumikawa et al Nat.Med. 2005

ABR wave patterns (24 kHz tone bursts)

Left (Atoh1 treated) Right

With permission, Raphael 2003

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Repeat ATOH1 Gene Therapy in different lab (Atkinson,et al PLOS, 2014)Results 1. ATOH1 treated resulted in increase in HC post injury2. ABR didn’t improve

Challenges for Gene Therapy in the Inner Ear

Depletion of important endogenous cell types (supporting cells, pillar cells, etc)

Electrical environment of hair cells/stria vascularis

Immune response Problems with viral vectors InnervationTiming

Molecular Therapy – Small molecule progenitor Cell Activation

Mitzutari et al (Neuron 2013) showed that they could use what they’d learned in a dish about genes important for cell fate (Notch, Sox, etc.) to inhibit a cell fate genetic signal after damage and turn on the transcription factor that stimulated Atoh 1 – this resulted in endogenous cells converting into hair cells in the damaged cochlea.

Video abstract: Neuron volume 77 Issue 1: 58-69

From Fuioka, Okano and Edge (2015) From Mizutari et al “Notch Inhibition Induces Cochlear Hair cell regeneration and recovery of hearing after acoustic trauma” 2013

Based on this work,Amsterdam–based Audion Therapeutics is developing a drug to inhibit Notch signaling. Eli Lilly, which developed the γ-secretaseinhibitor used by Edge, is supporting Audion’s program

Notch signaling declines in first post-natal week in mice

Maass et al 2015 Frontiers in Cellular Neuroscience

If Notch isn’t available how does blocking it work in adults?

Mitzutari et al 2013, Neuron

Maass et al 2015 Frontiers in Cellular Neuroscience

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Molecular Therapy - Small Molecule Progenitor Cell Activation – if Notch isn’t available can we activate an existing precursor?

Lgr5+ supporting cells in the cochlea give rise to hair cells in normal development (McLean et al 2016; 2017)

Molecular Therapy - Small Molecule Progenitor Cell Activation

Instead of forcing conversion of cells that don’t have ATO1 available, Lgr5 positive supporting cells through a Wnt/Notch pathway to proliferate (McLean et al 2016; 2017)

This could solve the challenge of cell depletion

Challenges for Molecular Therapy in the Inner Ear -

endogenous

Electrical environment of hair cells/stria vascularis

Innervation Timing ?

Depletion of important endogenous cell types (pillar cells, etc.)

Immune Response

Problems with viral vectors

From The Scientist, Sept. 1, 2015

Preclinical trials are feasibility and safety trials in animals

Clinical trial in humans delivering ATOH1

Novartis trial is currently (2014) recruiting participants (https://clinicaltrials.gov) The trial involves injecting a viral vector loaded with ATOH1 into the inner ear via a hole surgically drilled in the footplate of the stapes. The vector carries a promoter gene that confines ATOH1 expression to support cells of the cochlea.

Surgery has been performed on 6 patients with severe to profound hearing loss (University of Kansas Medical Center, Johns Hopkins School of Medicine, Columbia Med. School NY).*

Participants are now being monitored for changes in balance, hearing and neural activity in the central auditory pathway *

*according to The Scientist Sept. 1 2015

So translation from animal model –chick – to animal model mammal is progressing rapidly. We are working to overcome challenges presented to both exogenous approaches (stem cells) and endogenous approaches (gene therapy)

We know that, at least for birds where regeneration occurs naturally and within days/weeks of hair cell loss, auditory perception recovers or at least remains plastic and vocal production is only temporarily affected.

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What is the role of the Audiologist??

The basis for one of the clinical trials mentioned is that:

A. We can inject a genetic factor that will release inhibition for cell division in the cochlea

B. We can inject ATOH1 through a viral vector to cause remaining cells to convert to hair cells

C. We can inject stem cells through the round window and they will divide to become hair cells

We’ve come a long way in 29 years Where will we be in the next 29?

?

?

“The mind once expanded to the dimensions of larger ideas, never returns to its original size” Oliver W. Holmes

How does Cord Blood fit into the Stem Cell Picture?

Cord blood is a source of hematopoietic stem cells – cells that are multipotent

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How does Cord Blood fit into the Stem Cell Picture?

Clinical Trials

Safety of Autologous Human Umbilical Cord Blood Mononuclear Fraction to Treat Acquired Hearing Loss in Children – initiated 2011, Suspended 2014Safety of Autologous Stem Cell infusion for Children with Acquired hearing loss – initiated 2013, Suspended 2016

http://clinicaltrials.gov/ct2/show/record/NCT01343394

What is the Hair cell gene?

1. NOTCH2. WNT3. ATOH1

One Challenge for Gene Therapy is:

1. Depletion of endogenous cells in the cochlea

2. Integration of cells into the site of lesion

3. Cells tend to be driven to neural fate

Hair cells don’t naturally regenerate in the human cochlea because:

A. Cell division in the cochlea is under strict genetic inhibition

B. Morphogens and transcription factors during embryogenesis are not available in maturity

C. Both A and B are true

Stem Cell Therapy may be most successful:

A. To increase numbers of neuronsB. To increase numbers of endogenous support

cellsC. To increase numbers of hair cells

The basis for one of the clinical trials mentioned is that:

A. We can release genetic inhibition for cell division in the cochlea

B. We can inject ATOH1 through a viral vector to cause remaining cells to convert to hair cells

C. Lgr5 cells can be converted to hair cells in the living cochlea

Documents

AudNOWARC 2017a.ppt - Audiology ARC handout.pdf3/13/2017 1 BRENDA M. RYALS, PH.D. APRIL 5, 2017 ARC Disclosure I have relevant financial relationship(s) with the products or services