iPS-derived ocular cell exosomes as an injectable therapy for

glaucoma and age-related macular degeneration

Abstract
Glaucoma and age-related macular degeneration (AMD) are the leading causes of blindness in the United
States. The cause of these diseases is damage or degeneration of tissue within the eye, specifically the optic
nerve for glaucoma and the retina for AMD. Significant deterioration over time causes loss of function and
therefore blindness. Although these diseases impact millions of people in the US, there is currently no effective
treatment option to replace or restore the lost tissue. Both of these conditions could be made reversible
through tissue engineering applications, significantly reducing their global impact.

One therapeutic technique that shows great potential is the use of exosomes. Exosomes are vesicles released
from many cells that have been a growing interest of many studies for their role in disease. These vesicles play
an important role in intercellular communication through proteins, mRNA, microRNAs (miRNAs), and
cytokines. One protein carried by exosomes, myocilin, is found in lower amounts of individuals with eye
disease, suggesting that exosome-mediated delivery of proteins plays a role in preventing disease. In addition,
mutations in myocilin are present in many forms of glaucoma. While the role of myocilin and myocilin-
associated exosomes in eye disease has been studied and established, use of exosome-based therapies for
treatment of disease has not been established. Also, it is likely that exosome-associated RNA as well as other
proteins play significant roles in eye disease and thus require further study.

Our goal is to use induced pluripotent stem cell (iPSC)-derived ocular cell exosomes to prevent and repair
glaucoma and AMD. In performing this study, we will analyze the component profiles of ocular exosomes from
mice and iPSC-derived ocular cells in order to establish comparability between the two exosome sources. We
hypothesize that iPSC-derived ocular cell exosomes will contain critical components capable of
restoring ocular tissue after injection in mice with glaucoma and AMD.

To accomplish our aims, we will isolate exosomes from both healthy and diseased mice and perform RNA-seq
to determine the levels of critical exosomes components. We will use this information to identify specific RNAs
that are expressed at higher or lower levels than normal in the diseased state. Next, we will generate iPS-
derived ocular cell exosomes in vitro and determine whether these exosomes contain the appropriate amount
of critical RNAs to provide therapeutic benefits. Finally, we will inject iPS-derived ocular cell exosomes into
diseased mice and monitor their cellular function and regenerative capacity. This proposed research could
potentially lead to a therapeutic for resolving glaucoma and AMD. Additionally, this study could identify the
differences in critical exosome components that are lost in diseases tissues, allowing for therapies that
reintroduce the critical component.
Specific Aims
Glaucoma and age-related macular degeneration (AMD) are the leading causes of blindness in the United
States. These diseases contribute a significant amount to the $717 billion in spending in the U.S. that eye
disease is projected to cost by 2050 [1]. The cause of these diseases is damage or degeneration of tissue
within the eye, specifically the optic nerve for glaucoma and the retina for AMD. Significant deterioration over
time causes loss of function and therefore blindness. Although these diseases impact millions of people in the
US, there is currently no effective treatment option to replace or restore the lost tissue.

Exosomes are vesicles released from many cells that have been a growing interest of many studies for their
role in disease [2]. These vesicles play an important role in intercellular communication through proteins,
mRNA, microRNAs (miRNAs), and cytokines. One protein carried by exosomes, myocilin, is found in lower
amounts of individuals with eye disease, suggesting that exosome-mediated delivery of proteins plays a role in
preventing disease [3]. In addition, mutations in myocilin are present in many forms of glaucoma. While the role
of myocilin and myocilin-associated exosomes in eye disease has been studied and established, use of
exosome-based therapies for treatment of disease has not been established. Also, it is likely that exosome-
associated RNA as well as other proteins play significant roles in eye disease and thus require further study.

Our aim is to use induced pluripotent stem cell (iPSC)-derived ocular cell exosomes to prevent and repair
glaucoma and AMD. In performing this study, we will analyze the component profiles of ocular exosomes from
mice and iPSC-derived ocular cells in order to establish comparability between the two exosome sources. We
hypothesize that iPSC-derived ocular cell exosomes will contain critical components capable of
restoring ocular tissue after injection in mice with glaucoma and AMD.

This hypothesis will be addressed in the following Specific Aims:

Specific Aim 1: To establish mouse disease models using mice as an animal models. Induce eye disease
(glaucoma and AMD) in two group of mice, while the third group without eye diseases is a control group.

Specific Aim 2: To compare iPSC-derived ocular cell exosomes to mouse disease-model exosomes in
order to understand any differences in exosome presence. We will differentiate iPSC into ocular cells. Next, the
exosomes present in each scenario will be quantified in order to enable a straightforward comparison. Protein
profiles of each will be examined and compared to infer a possible exosome role in glaucoma and other AMDs.

Specific Aim 3: To inject iPSC-derived ocular cell exosomes into mouse eye with disease model.
We assume that the lower amount of myocilin and its mutation is related to eye diseases, therefore by injecting
the iPSC-derived ocular cell exosomes into the eye of mice with eye disease, we anticipate that the mice will
have expression of the exosomes and therefore the eye diseases will be alleviated. To test the severity of
glaucoma in mice, we will use tonometer to measure the pressure inside the eye, and use pachymetry to
measure the thickness of the cornea. To test the severity of AMD in mice, we will use fluorescein angiogram to
discover broken vessels in the eye, and use optical coherence tomography to get the image of retina.
Research Strategy

A. Significance

Eye diseases, chiefly age-related macular degeneration (AMD) and glaucoma, pose a serious problem to the
aging population. Glaucoma is an incurable degenerative eye disease affecting over 60 million people
worldwide with projections reaching nearly 80 million by 2020 [1]. Of these cases, it is estimated that only half
know they have glaucoma [2]. If not treated early-on, glaucoma leads to irreversible blindness and even in
those who receive timely, proper treatment, 10% still develop sight loss. Age-related macular degeneration will
affect over 196 million worldwide by 2020 [3]. The most effective therapy for AMD is the use of anti-VEGF
drugs which merely slow the progression of the disease. There are no cures to regain lost and damaged
tissue. Both of these conditions could be made reversible through tissue engineering applications, significantly
reducing their global impact.

Recent research shows that exosomes, small vesicles previously thought to only serve in cellular waste
disposal, play a significant role in intercellular signaling by transporting RNAs and proteins. Upon fusion with
another cell, an exosome delivers its contents causing physiological changes in the recipient cell due to the
resultant change in the RNA and protein profile. Exosome-based therapy is advantageous to cell-based
therapy in that it allows delivery of RNA and proteins while avoiding the immune-based complications of
transplanting cells. Previous study has shown that exosome-based therapies improve cardiac regeneration and
facilitate protection of local cells [5]. In addition, introduction of mesenchymal stem cell-derived exosomes
promotes the survival and regeneration of retinal ganglion cells reducing the effects of glaucoma [6].

It is well established that the eye undergoes numerous physiological changes through aging, many of which
are accelerated in cases of glaucoma and AMD [8]. Many of these changes occur in the retinal pigment
epithelium (RPE), a set of pigmented cells that maintain photoreceptor health and function. RPE cells are
known to release exosomes which likely play a role in the maintenance of normal photoreceptor physiology.
Isolation and characterization of RPE-derived exosomes from healthy and diseased models, including RPE
exosomes from iPSC-derived RPE cells will shed light on differences in exosomal makeup that lead to
degenerative disease.

In addition, the previously-demonstrated potential for exosomes to facilitate not only protection, but
regeneration, provides a clear route from exosome characterization to their application in therapies.
Additionally, studies have found that mutations in the gene GLC1A, which encodes a protein myocilin, are
present at a significantly higher rate in glaucoma patients than in the healthy population [11]. Efforts to
characterize this protein found that myocilin is an exosome-associated protein which supports an exosomal
analysis approach for glaucoma therapy [4]. It is likely that comparative study of healthy and diseased
exosome populations will help identify numerous other gene defects associated with eye disease. Identification
of genetic defects and correction through exosome-mediated introduction of healthy genes (from healthy cells)
is a promising area of study in developing cures to glaucoma and AMD.

The current proposal is motivated by the potential regenerative capacity of exosomes within the eye. We will
quantitatively analyze iPS-derived ocular cells and diseased ocular cells to establish differences in
critical exosome components and utilize iPS-derived ocular cells as an injectable therapeutic for
glaucoma and age-related macular degeneration. The results from this experiment are expected to provide
insight into future therapies for glaucoma and AMD using exosomal tissue engineering as a basis for protection
and regeneration of damaged tissue.
 B. Innovation
Previous studies have separately indicated that exosomes contain therapeutic potential for many diseases and
that cell therapies are not sufficient for the treatment of glaucoma and AMD. However, studies have not yet
indicated whether iPSC-derived ocular cells are capable of restoring function upon injection into mice with
glaucoma or AMD. This study will provide insight into the therapeutic potential of iPS-derived ocular exosomes.
This study is innovative in three respects: 1) quantification of RNA levels in diseased ocular cell exosomes,
2) quantification of RNA levels in iPSC-derived ocular cells as a comparison to healthy ocular cells from mice,
3) injection of exosomes into the eye as a regenerative therapy.

C. Proposed Research Approach

Specific Aim 1: To establish mouse disease model using mice as an animal models. Induce eye disease
(glaucoma and AMD) in two group of mice, while the third group without eye diseases is a control group.

Aim 1.1: Establish mouse AMD model.

For disease model of AMD, we are going to use a method described by J.G. Hollyfield et al in 2008
[12]. We will induce AMD-like lesions in mice by immunizing them with mouse serum albumin adduced
with carboxyethylpyrrole, which is previously found in adducting proteins in drusen from AMD donor eye
tissues [13] and in plasma samples in plasma samples [14] from individuals with AMD. Antibodies are
generated in immunized mice, and the binding causes an accumulation of drusen below the retinal
pigment epithelium, which is a mimic of geographic atrophy in AMD [12].

Interpretation of Results
To determine whether a mouse has developed AMD, we will use optical coherence tomography in order
to get the image of retina and the RPEBruchs membrane interface. The result from the AMD model
and the control group will be compared and quantified: including the thickness of the basal lamina of
the choriocapillaris, the thickness of Bruchs membrane, and the height of the basal infoldings.

Aim 1.2: Establish mouse glaucoma model

To study glaucoma, several rodent models were summarized by I. H. Pang et al in 2007 [15], including
both non-pressure models and pressure models, the former are intended to be surrogate models
designed to address certain specific aspects of the disease progress. To better mimic the
pathophysiological process of glaucoma in vivo, we hereby choose a pressure model. We will induce
glaucoma in mice by using laser to occlude the outflow pathway. In the mouse, laser damage to the
limbus or laser treatment after indocyanine green injection into the anterior segment can produce ocular
hypertension for up to 6 weeks [16][17][18]. Which will cause the loss of retinal ganglion cells and
thinning of all retinal layer as well as the mass degeneration of optic nerve axons, which are the same
symptoms appear in glaucoma.

Interpretation of Results
To determine whether a mouse has developed glaucomatous damage, morphologic, biochemical, and
functional assays have been developed to examine the pathological changes in the retina. However,
each of the techniques addresses only a limited and therefore incomplete aspect of the biologic
changes. Ideally, more than 1 technique should be used to provide a robust appraisal and interpretation
of study results [15].

Potential Pitfalls
 Although laser photocoagulation can mimic the characteristics of glaucoma, it is hard to perform. Sixty
to 80 laser burns are usually required around the circumference of the limbus. But even though treated
with the same manner with high accuracy, the duration and magnitude of the intraocular pressure in
each eye might varies. And the increased intraocular pressure cannot last for a long time after the
treatment, it will return to normal value in several weeks.

Alternative Approaches
To increase the reproducibility of high intraocular pressure, another pressure model can be an
alternative choice: episcleral injection of hypertonic saline. However, this approach has a higher
technical difficulty, especially on small animal model as mice. To use this approach, perhaps using rats
will be a better choice.

Specific Aim 2: Compare iPSC-derived ocular cell exosomes to mouse disease-model exosomes in
order to understand any differences in exosome presence.

Aim 2.1: Differentiate mouse iPSC into retinal cells.

Because both Glaucoma and AMD degenerate the neuroretinal cells of the eye [7], mouse iPSCs will
be differentiated into Retinal Ganglion Cells (RGC) and nourishing Retinal Pigment Epithelium (RPE) to
examine exosome content in each group. RPE cells were included due to their role in formation of ECM
and many metabolic pathways along with maintaining homeostasis of the retina [19]. Mouse iPSCs will
be induced from mouse embryonic fibroblasts by retroviral transfection as described by Parameswaran
et al. [20]. Embryoid bodies derived from these iPSCs will be cultured, trypsinized, expanded, and then
put in a retinal culture medium (RCM) for the differentiation phase to produce RGCs [20]. RPEs will be
produced from iPSC derived retinal progenitor cells following the Differentiation/Purification Protocol
for Retinal Pigment Epithelium put forth by Iwasaki et al. [21].

Aim 2.2: Isolate and extract exosomes from differentiated ocular cells and mice disease-model
ocular cells.
In order to quantify exosome presence in each cell type and sequence RNA, exosomes will be isolated
from culture or extracted from mice ocular cells and then isolated. Exosomes from induced cells will be
isolated from cell-culture media using centrifugation and ultracentrifugation steps, following the protocol
followed in Biasutto et al. [9]. Induced RGC and RPE originated exosomes will be precipitated out of the
media, washed, and then stored at -80C in an extraction buffer before preparation of microarray
evaluation [9]. Exosomes from the mice disease-model retinal cells will be more difficult to extract due
to the added steps of extraction from the animal. A protocol given in Barres et al. will be followed for
extraction and dissection of mice retina [10]. After enucleations and dissection, retinal cell suspensions
will be created with purity determined by immunofluorescence staining [10]. RGC will be isolated from
the cell-suspension by the panning procedure introduced by Barres et al. [10]. RPE cells will be
extracted from the mice using a RPE cell isolation by the mechanical dissociation method employed by
Wang et al. [19]. Exosomes from the mouse disease model-will then be isolated as per the protocol
followed by induced cells as stated previously.

Aim 2.3: Sequence RNA of exosomes from each source to compare disease-model to induced
cells. In order to run RNA-seq, steps need to be taken to isolate RNA from the surrounding exosome
content. This will be done using the Total Exosome RNA and Protein Isolation Kit from Invitrogen, as
per protocol followed by Schagamen 2013 et al. [26].

RNA libraries will be prepared and sequenced using Ion Total RNA-Seq Kit v2 protocol from Life
Technologies, as done by Schagamen 2013 et al. [26].
 The results from the RNA-sequencing from exosomes from all the above sources will be compared
against the known mouse transcriptome. We will quantify how many RNAs code for specific genes in
each group of exosomes that was analyzed. It is our hope that we will see clear trends differentiating
exosomal RNA originating from induced ocular cells versus mouse disease-model cells that will
elucidate possible exosomal roles in Glaucoma and AMD, and create a pathway for future steps in
therapies to treat these degenerative diseases.

Potential pitfalls are that exosomes play many roles and could vary from exosome to exosome, so
determining whether an extracted exosome is representative of the entire exosome population will
prove difficult. In order to decrease the likelihood of this variation, we will make sure comparisons
between the two exosome types are statistically significant, not singular occurrences.

Specific Aim 3: To inject iPSC-derived ocular cell exosomes into mouse eye with disease model.
To test if the iPSC-derived ocular cell exosomes injection can be an effective treatment for AMD and glaucoma,
we will inject iPSC-derived ocular cell exosomes into diseased mice model and do analyze.

Aim 3.1: Inject iPSC-derived ocular cell exosomes into diseased mice model.
Mouse model of AMD and mouse model of glaucoma are established as the description in Aim 1.1 and
Aim 1.2. iPSC-RGC exosomes and iPSC-RPE exosomes are prepared as description in Aim 2.2. The
experiments are designed as followed. 30 Mice were divided into following 5 groups: Group 1, healthy
mice; Group 2, AMD mice/iPSC-RGC exosomes; Group 3, AMD mice/iPSC-RPE exosomes; Group 4,
Glaucoma mice/iPSC-RGC exosomes; Group 5, Glaucoma mice/iPSC-RPE exosomes. Only right eyes
of animals were used and left eyes were regarded as normal controls. Refer to the frequency of
injection applied by Mead, B et al in 2017 [6], we choose to inject the exosomes on day 0, 7 and 14 and
animals were sacrificed on week 6. We will label the exosomes with red fluorescent dye using a
commercially available kit according to the instruction.

Interpretation of Results
As we labeled the exosomes, if the exosomes are successfully injected, they can be observed by
fluorescence microscopy and we can get the permeability and dynamic distribution of exosomes in mice
eye. The animals will live through experiment without having adverse reaction.

Potential Pitfalls and Alternative Approaches

As we do not know all the functions of the two kinds of exosomes, there may exists unpredictable
questions after injection. We can adjust the concentration of the exosomes, total amount of exosomes, or
the frequency of injection to optimize the treatment.

Aim 3.2: Assessments of therapeutic potential of iPSC-derived ocular cell exosomes.

Exosomes from different cells have different functions. Exosomes released from RACs containing
antiangiogenic components that might protect the eye from angiogenesis and maintain its functional
integrity. [22] However, exosomes obtained from Akt-modified hucMSCs are effective in myocardial
infarction therapy through promoting angiogenesis. [23]. To assess the therapeutic potential and study
the functions of iPSC-RGC exosomes and iPSC-RPE exosomes, several measurements will be used.
Intraocular pressure (IOP) is an important index of glaucoma, IOP will be measured as previously
described. [24] AMD is characterized by the abnormal growth of new blood vessels within or beneath
the macula, we will use fluorescein angiogram to analyze the vessels in the eye. In addition,
electroretinography (ERG) was used as a measure of rat visual function and optical coherence
tomography (OCT) will be used to take the images of the retina and quantified the thickness.
 Histological analysis will be done according to the method described previously [25] All experiments
were repeated at least two times. Statistical analysis was performed using the Students t-test for two
sets of data.

Interpretation of Results
To determine whether the exosomes have the therapeutic potential, many assays will be done and the
functions of two exosomes will be compared. We anticipate that the mice will have expression of the
exosomes and therefore both two kinds of the eye diseases will be alleviated based on the results well
get through the measurements. In addition, we will unite the functions and RNA information we get in
Aim 2.3 to analyze and compare the exosomes we get.

Potential Pitfalls and Alternative Approaches

However, one possibility exists that the exosomes have no or little effects. After getting the functions of
exosomes, we can try to use a mixture of different kinds of exosomes, by adjusting the ratio between
various composition, we expect to get a better treatment effect.

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