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In: The CRISPR/Cas9 System ISBN: 978-1-53616-426-8

Editor: Alfred A. Bertelsen © 2019 Nova Science Publishers, Inc.

Chapter 3

THE APPLICATION OF CRISPR/CAS9

THERAPIES IN OPHTHALMOLOGY AND

RECENT ADVANCES FOR THE TREATMENT

OF GENETIC EYE DISEASE

C. B. Tara Moore1,, PhD, Larry A. DeDionisio2,†, PhD,

Hila Roshanravan2,‡, PhD, Connie Chao-Shern2,§

and M. Andrew Nesbit1, #, PhD 1Ulster University, Coleraine, County Londonderry,

UK/Northern Ireland 2Avellino Lab USA, Menlo Park, CA, US

Corresponding Author’s E-mail: [email protected]. † Author’s E-mail: [email protected]. ‡ Author’s E-mail: [email protected]. § Author’s Email: [email protected]. # Author’s E-mail: [email protected].

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C. B. Tara Moore, Larry A. DeDionisio, Hila Roshanravan et al. 100

ABSTRACT

Clustered regularly interspaced short palindromic repeats (CRISPR)

and CRISPR-associated (Cas) protein systems have been used to target

chosen regions of the genome with high efficiency and specificity and,

together with the multiplexing capabilities of this technology, have

introduced a novel tool to the field of genome engineering. With a potential

for the permanent treatment of genetic ocular diseases, CRISPR based

therapeutics hold great potential for the field of ophthalmology. CRISPR

systems can repair genetic mutations in recessive diseases where both

copies of a gene carry a pathogenic mutation, or they can knock out the

pathogenic gene allele in dominant diseases where only one copy of the

gene carries the mutation leaving the functionality of the second gene copy

in place. The majority of corneal dystrophies are the result an autosomal

dominant inheritance pattern within the TGFBI gene, which presents an

ideal model suited for a CRISPR/Cas knock out methodology. This

approach can also be applied to the treatment of autosomal dominantly

inherited genetic disease in general. There are several ocular diseases that

are currently being investigated worldwide for which human gene transfer

trials have been approved. This review summarises the use of CRISPR-Cas

gene editing in ophthalmology and focuses on the advancement of gene

editing in the cornea.

1. INTRODUCTION

Vision is often considered the most important human sense. Our eyes

facilitate daily activities from basic mobility to the appreciation of art and

nature. Genetically heritable eye disease have the potential to impact those

who suffer from them in profound ways. In ophthalmology, disease-causing

genetic mutations that lead to retinal disease and the various types of corneal

dystrophy (CD) often display directly observable ocular manifestations to

the clinician. However, the considerable heterogeneity of inherited ocular

disease presents difficulty in choosing a treatment plan. Many of the

inherited, non-syndromic eye diseases are caused by the very specific spatial

expression of the mutated genes (Tucker, Mullins, and Stone 2014). To date,

more than 3000 genetic mutations in approximately 70 genes have been

associated with retinitis pigmentosa (RP) (Dias et al. 2018) and at least 8

The Application of CRISPR/Cas9 Therapies in Ophthalmology … 101

genes have been identified for diverse corneal CDs (Klintworth 2009,

Schorderet 2015). The International Committee of the Classification of

Corneal Dytrophies (IC3D) outlined genetic links to 22 different types of

CD (Weiss et al. 2015). A precision or personalised medicine that functions

to identify the genetic basis of a disease in the patient and treat it with a

tailored approach is the most effective way to treat these types of maladies.

Gene therapy is defined as either the introduction of exogenous DNA or

RNA into a patient's cells or the silencing of pathogenic gene mutations

within the patient’s DNA. Silencing endogenous gene mutations known as

gene editing is achieved either by gene knockout or mutation correction.

Genetics based therapeutic approaches include RNA interference (RNAi)

with short interfering RNA (siRNA), gene replacement and gene editing via

programmable nucleases. Gene replacement therapies, where a functional

copy of the gene to treat loss of function mutations commonly introduced

via viral transgene expression have translated to clinical trials for inherited

ocular disease of the retina. These therapies treat autosomal recessive

conditions (Parker et al. 2016, Weleber et al. 2016, Hauswirth et al. 2008,

Bainbridge et al. 2008, Bennett et al. 2016, Ghazi et al. 2016, Edwards et al.

2016, Michalakis et al. 2017, Dalkara et al. 2016). In this context, biallelic

pathogenic mutations in a disease-associated gene could be supplemented

with the wild type, functional version of the gene. However, this strategy

cannot be applied to dominant conditions, where the pathogenic gene needs

to be repaired or down-regulated. The Moore research group at Ulster

University has extensively studied the application of siRNA for treatment of

autosomal dominantly inherited genetic diseases of the skin and eye and

have clearly demonstrated the potential of therapeutic siRNA for the cornea

(Courtney, Atkinson, Allen, Moore, Walsh, Pedrioli, MacEwen, Pellegrini,

Maurizi, Serafini, et al. 2014, Courtney, Atkinson, Moore, et al. 2014, Allen

et al. 2013, Liao et al. 2011, McLean and Moore 2011, Atkinson et al. 2011,

Moore et al. 2011).

For clinical treatment of autosomal dominant diseases, gene editing

holds enormous potential. Gene editing makes use of programmable

nucleases to target DNA in a sequence specific manner inducing a double

strand break (DSB). The DSB allows the mutation within the DNA sequence


to either be removed by non-homologous end-joining (NHEJ) or repaired

via homology directed repair (HDR) (Rudin, Sugarman, and Haber 1989,

Plessis et al. 1992, Rouet, Smih, and Jasin 1994, Choulika et al. 1995,

Bibikova et al. 2001, Bibikova et al. 2003, Bibikova et al. 2002). Four

classes of programmable nucleases are currently used, meganucleases,

(Belfort and Bonocora 2014, Stoddard 2011) zinc finger nucleases (ZFNs),

(Urnov et al. 2010) transcription activator-like effector nucleases (TALENs)

(Bogdanove and Voytas 2011) and clustered regularly interspersed

palindromic repeats (CRISPR) associated nuclease, Cas9 (Jinek et al. 2012,

Cong et al. 2013, Mali, Yang, et al. 2013, Zetsche et al. 2015). CRISPR/Cas

systems are derived from an endogenous defence mechanism identified

within a diverse group of bacteria and archaea (Karginov and Hannon 2010,

Makarova et al. 2011, Makarova et al. 2015). Compared with the extensive

protein engineering required for the meganucleases and ZFNs,

CRISPR/Cas9 can be easily programmed with simple molecular cloning

techniques. Modifications have been engineered to enhance the

CRISPR/Cas9 complex for genome editing within mammalian cells

facilitating the use of CRISPR/CAS9 to treat various human diseases (Xu et

al. 2015, Schwank et al. 2013, Lee et al. 2015).

The ease of using the CRISPR system has resulted in it rapidly replacing

other programmable nucleases as the preferred tool for genome editing.

Gene editing with CRISPR/Cas9 also largely circumvents the limit of viral

vector capacity faced by gene-replacement therapies (Ran et al. 2015, Zuris

et al. 2015). This efficient technology is particularly attractive in

ophthalmology. The eye’s accessible anatomical position, immune privilege

and the blood-retinal barrier make it an ideal site for in vivo gene editing

(Liu, Tuo, and Chan 2011). It is foreseen that CRISPR/Cas based therapy

for ocular disease will pave the way for personalised medicine. This review

discusses the progress in the use of CRISPR/Cas systems for treating eye

disease with an emphasis on recent developments in the treatment of rare

and inherited disorders of the cornea.


2. APPROACHES FOR GENE EDITING WITH CRISPR/CAS

Cas9 is a crucial component in the type II CRISPR/Cas system and

cleaves double-stranded DNA (dsDNA) in a sequence directed and specific

manner. The first step in this protein-DNA interaction is binding of the Cas9

nuclease to the DNA target site via a single guide (sg) RNA sequence that is

complementary to the target DNA and to a short segment of DNA adjacent

to the target region referred to as the protospacer adjacent motif (PAM).

Figure 1. Cas9 programmed by tracrRNA:crRNA duplex.

The PAM acts as a recognition site for Cas9 and is critical in its ability to

cleave the target DNA (Figure 1). Alteration of the 20 nucleotide spacer

sequence within the sgRNA, lying directly upstream of the PAM directs the

RNA-guided endonuclease to the desired target sequence (Jinek et al. 2012,

Xu et al. 2014). Different PAMs are recognised by Cas nucleases from

different bacterial species. The most characterised type II systems are S.


pyogenes Cas9, which recognises a 5–NGG–3′ PAM and S. aureus Cas9,

which recognises a 5–NNGRRT– 3’ PAM (Belfort and Bonocora 2014,

Mojica et al. 2009). The PAM-proximal region of the guide, often referred

to as the ‘seed region’ (Semenova et al. 2011) dictates the binding of Cas9

to the target site so mismatches in this region are critical in determining

specificity while mismatches in the distal region have less impact on the

specificity (Pattanayak et al. 2013, Sternberg et al. 2014, Wu et al. 2014).

There is no uniformity in the exact length of the seed region; however, the

studies suggest it is the first 5-12bp of the guide sequence that is critical.

2.1. Minimising off Target Cleavage and Improving Efficiency

There are limitations that can hinder applications of the CRISPR/Cas9

system such as inefficient delivery, dysregulation of the delivered gene, the

immune response against the CRISPR system, and off-target effects or the

unintended on-target mutations at sites that share identical or highly similar

sequence to the target locus (Cho et al. 2014, Fu et al. 2013, Hsu et al. 2013,

Pattanayak et al. 2013, Lin, Cradick, et al. 2014, Lee and Kim 2019). There

are studies that determined the mutation frequency in CRISPR/Cas9 edited

mouse and monkey models were not significantly higher than in wild type

control animals (Willi et al. 2018, Luo et al. 2018). These studies did not

rule out the possibility of rare mutations at genomic loci that possess

sequence homogeneity to the target sequence. To counteract these unwanted

genomic editing events, the sequence characteristics of the sgRNA, which

are critical for improving specificity and efficiency should be carefully

monitored. Guides with high cleavage efficiency are necessary within a

clinical setting in order to achieve therapeutic benefits. Several online tools

have been designed to allow in silico analyses of on- and off-target cleavage

of each guide sequence. Studies that utilized optimal guide design in

conjunction with genome-wide analyses have reported minimal off target

cleavage (Lin, Cradick, et al. 2014, Kim et al. 2015, Veres et al. 2014, Frock

et al. 2015, Tsai et al. 2017).


Table 1. Different techniques to identify off-targets

Methods Description

linear amplification–mediated

high-throughput genome-wide

sequencing (LAM-HTGTS)

High coverage sequencing to assess the degree of

mutagenesis across the entire genome (Hu et al. 2016)

Di-genome seq in vitro Cas9-digested whole-genome sequencing (Kim et

al. 2015)

Circularization for in vitro

reporting of cleavage effects

by sequencing (CIRCLE-seq)

A highly sensitive, sequencing-efficient in vitro screening

strategy for identifying CRISPR-Cas9 genome-wide off-

target mutations (Tsai et al. 2017)

Breaks Labelling, Enrichment

on Streptavidin, and

Sequencing (BLESS)

The first method for direct labelling of DSBs in situ

followed by their genome-wide mapping at nucleotide

resolution (Crosetto et al. 2013)

Breaks Labelling In Situ and

Sequencing (BLISS)

1) Direct labelling of DSBs in fixed cells or tissue

sections on a solid surface

2) low-input requirement by linear amplification of

tagged DSBs by in vitro transcription

3) quantification of DSBs through unique molecular

identifiers

4) easy scalability and multiplexing (Yan et al. 2017)

Genome-wide Unbiased

Identification of DSBs

Enabled by Sequencing

(GUIDE-Seq)

Relies on capture of double-stranded

oligodeoxynucleotides into breaks (Tsai et al. 2015)

SITE-Seq A biochemical method, based on the selective enrichment

and identification of adapter-tagged DNA ends by

sequencing (Cameron et al. 2017)

Several methods have been devised to directly identify cleaved sites in

cells (Table 1) and these techniques have achieved good sensitivity and

resolution. However, at the present time there is no single “gold standard

method” which can accomplish unbiased, comprehensive off-target

analysis. To reduce the off-target activity of CRISPR/Cas, much focus has

been directed on improving target specificity of the system. Several

strategies to enhance Cas9 specificity have been reported (Table 2).


Table 2. Different strategies to enhance Cas9 specificity

Techniques Description

Reducing the amount of

active Cas9 in cells

Studies have shown increased off-target activities with high

levels of Cas9 protein (Fu et al. 2013, Hsu et al. 2013, Pattanayak

et al. 2013) and a number of techniques have been employed to

restrict the concentration of Cas9 protein such as delivering it as

a purified protein loaded with sgRNA (Cho et al. 2013, Kim et al.

2014) or limiting the duration and expression of Cas9, using

various inducible platforms (Davis et al. 2015, Zetsche, Volz,

and Zhang 2015).

Double nicking by Cas9

nickase mutants

Creates a pair of juxtaposed single-stranded DNA nicks to

enhance genome editing specificity (Ran et al. 2013, Mali, Aach,

et al. 2013).

Altering the sgRNA

sequence

Several studies showed that truncation of sgRNA leads to an

increased target specificity and reduced off target activity.

(Pattanayak et al. 2013, Fu, Reyon, and Joung 2014, Fu et al.

2014) However, it has been shown by our group that a range of

truncated guide lengths or addition of 5′-GG to the 20 nt guide

sequence did not provide a marked improvement of specificity

(Figure 2) (Christie et al. 2017).

Fusion of catalytically

inactive Cas9 to FokI

nuclease (fCas9)

Target DNA sites modified in human cells with >140-fold

higher specificity than wild-type Cas9 and the specificity of

fCas9 was around 4-fold higher than that of paired nickases at

loci with highly similar off-target sites (Guilinger, Thompson,

and Liu 2014, Tsai et al. 2014). More recent developments have

incorporated the Fokl-Cas9 system with truncated sgRNA to

further improve specificity and reduce off-target activities

(Wyvekens et al. 2015). Although each of these approaches

reduce off-target mutagenesis, they have a number of limitations:

reducing the amount of Cas9 can decrease on-target cleavage

efficiency and truncated guides can increase indel formation at

some off-target loci.

Improve the specificity

of Streptococcus

pyogenes Cas9

(SpCas9) is based on

structure-guided protein

engineering.

A study reported that neutralization of positive charges in the

non-target groove can dramatically decrease off-target indel

formation while preserving on-target activity (Slaymaker et al.

2016). In another report, engineering of additional high-fidelity

variants of CRISPR-associated nucleases by introducing

substitutions at additional non-specific DNA contacting residues

can further reduce some of the very small number of residual off-

target sites that persist for certain sgRNAs (Kleinstiver et al.

2016).


In addition to the work carried out to reduce off-target effects, much

effort has been done to enhance the gene editing efficiency of the

CRISPR/Cas9 complex. The DSBs introduced by the enzyme are most

commonly repaired by the NHEJ pathway, leading to nonspecific nucleotide

insertions and deletions, referred to as ‘indels’.

This is convenient for generating gene knockouts but does not allow

introduction of specific sequence changes. The alternative path to repair,

HDR enables a recombination event between a homologous donor DNA

template and damaged DNA resulting in an accurate repair of the DSB.

Many groups have shown different strategies to improve the HDR rate of

repair over the NHEJ pathway (Zhang et al. 2017, Paquet et al. 2016, Kwart

et al. 2017, Liang et al. 2017). For example, the inhibition of DNA ligase

IV, the key enzyme utilized by NHEJ with the inhibitor Scr7 can be used to

promote HDR (Maruyama et al. 2015, Chu et al. 2015, Singh, Schimenti,

and Bolcun-Filas 2015). HDR is restricted to the G2 phase of cell division

when DNA replication is completed and sister chromatids are available to

serve as repair templates, (Lin, Staahl, et al. 2014) which limits the use of

this pathways in certain cell types such as ocular cell types where the cells

are primarily in the post-mitotic G0 state (Young 1985).

2.2. Expanding the CRISPR/Cas Protospacer Adjacent Motif

(PAM) Repertoire

Cas9 nucleases from various bacterial species have been shown to have

different PAM sequences (Table 3) including a newly characterised member

of the CRISPR programmable nuclease system, Cpf1 from the

Acidaminococcus and Lachnospiraceae bacteria, which was adapted for

genome editing in human cells (Zetsche et al. 2015). Its PAM motif, TTTN

was shown to provide the best cleavage efficiency. The expanding

CRISPR/Cas PAM repertoire will allow greater targeting across diverse

genomic regions. Moreover, it has been reported that artificially engineered

SpCas9 and SaCas9 with alternative PAM recognition motifs can be

generated (Kleinstiver, Prew, Tsai, Nguyen, et al. 2015, Kleinstiver, Prew,


Tsai, Topkar, et al. 2015). Thus, the application of directed evolution

technology to the development of Cas9 with varying PAM motifs will

expand the repertoire of targetable sequences throughout the human genome

and increase the range of accessible therapeutic targets.

Table 3. CRISPR/Cas Protospacer Adjacent Motif (PAM) repertoire

Source Nuclease type PAM sequence

Streptococcus

pyogenes

Cas9 NGG(Jinek et al. 2012)

Cas9 (D1135E) with

improved recognition and

speficity

NGG with (Kleinstiver, Prew,

Tsai, Topkar, et al. 2015)

Cas9 (“VQR”;

D1135V/R1335Q/T1337R)

NGAN or NGNG (Kleinstiver,

Prew, Tsai, Topkar, et al. 2015)

Cas9 (“EQR”;

D1135E/R1335Q/T1337R)

NGAG (Kleinstiver, Prew, Tsai,

Topkar, et al. 2015)

Staphylococcus aureus Cas9 NNGRRT or NNGRR(N) (Plessis

et al. 1992)

Cas9 (“KKH”;

E782K/N968K/R1015H)

NNNRRT (Kleinstiver, Prew,

Tsai, Nguyen, et al. 2015)

Neisseria meningitides Cas9 NNNNGATT (Hou et al. 2013)

Streptococcus

thermophiles

Cas9 NNAGAAW (Sapranauskas et al.

2011)

Francisella novicida Cpf1 TTN (Zetsche et al. 2015)

Acidaminococcus sp.

BV3L6

Cpf1 TTTN (Zetsche et al. 2015)

Lachnospiraceae

bacterium

Cpf1 TTTN (Zetsche et al. 2015)

Alicyclobacillus

acidoterrestris

C2c1 TTN (Shmakov et al. 2015)

IUPAC nucleotide code, where R refers to A or G; W to A or T; and N represents any nucleotide.

3. CRISPR-CAS9 GENE EDITING IN GENETIC

EYE DISEASE

The eye is an ideal site for testing of in vivo gene editing due to its

accessible position, compartmentalised anatomy and relative immune-

privilege. CRISPR-Cas9-based therapeutics provide the possibility for


permanent treatment of genetic disease. Inherited eye diseases affect

millions of people worldwide and have an immense socioeconomic impact.

With our increasing understanding of the underlying molecular mechanisms

of ocular diseases, gene therapy, especially combined with adeno-associated

viruses (AAV) delivery, has been proposed as an effective approach. With

the advantages of CRISPR/Cas over alternate genome editing technologies,

the stage is set for a new wave of gene-based clinical trials. The benefits of

using CRISPR/Cas9 systems to study eye diseases have been highlighted by

several successful applications in a variety of cell types and animal species.

3.1. Applications for Retinal Eye Disease

Research into CRISPR/CAS9 gene editing as applied to ocular diseases

was initially focused on diseases that affect the retina. Hereditary retinal

diseases comprise the leading cause of blindness among working age adults

in the United Kingdom (Liew, Michaelides, and Bunce 2014). Retinal

diseases like proliferative diabetic retinopathy (PDR), retinopathy of

prematurity (ROP), and wet age-related macular degeneration (wAMD) are

known to occur from abnormal angiogenesis, the process by which new

blood vessels grow from pre-existing vessels (Abhinand et al. 2016).

Vascular endothelial growth factor (VEGFR) plays a critical role in

angiogenesis, and experiments utilizing CRISPR/Cas9 to edit VEGFR have

shown promise. Editing VEGFR2 in mouse models for oxygen-induced

retinopathy (OIR) and laser-induced choroid neovascularization (CNV) was

shown to abrogate angiogenesis (Huang et al. 2017). In addition, another

study showed the knockout of Vegfa and Hif1a genes by CRISPR-Cas9 to

block angiogenesis in a CNV model in mouse (Kim, Park, et al. 2017).

Subretinal injection of sgRNA- ribonucleoproteins (RNPs) complexes into

the adult mouse eye gave rise to mutagenesis at the target Vegfa gene in the

retinal pigment epithelium resulting in reduced CNV in an AMD mouse

model (Kim, Park, et al. 2017).

Targeted genomic deletion using the CRISPR/Cas9 system presents a

promising therapeutic approach for the treatment of patients with Leber's


congenital amaurosis 10 (LCA10) bearing the CEP290 splice mutation

(Ruan et al. 2017b). Loss of cone photoreceptors in RP leads to blindness,

and preservation of cone function is a major therapeutic goal. Sub-retinal

injection of AAV-delivered CRISPR/Cas9 prevented retinal degeneration in

mice by Nrl knockdown in rod cells (Yu et al. 2017). Furthermore, gene

editing using CRISPR/Cas9 has been shown to correct retinal dystrophy in

a rat model of autosomal dominant RP by generating allele-specific

disruption of RhoS334 (Bakondi et al. 2016).

3.2. Corneal Autosomal Dominant Genetic Eye Disease–

Allele Specificity

The majority of corneal dystrophies are the result of an autosomal

dominant inheritance pattern (Klintworth 2009). Others exhibit autosomal

recessive inheritance, while a few corneal dystrophies present X-linked

inheritance. To date, 23 distinct corneal dystrophies have been described.

Table 4 describes each corneal dystrophy, including; associated inheritance

pattern, gene locus and causative genes. Corneal dystrophies are

predominantly monogenic and highly penetrant. The accessibility of the

cornea, make the corneal dystrophies an ideal starting point for genome

editing therapies where concepts of personalized medicine can be put to use

(Moore et al. 2018).

Research into the use of CRISPR/Cas9 for the treatment of rare inherited

corneal disease has progressed during the past few years. We were the first

to demonstrate an allele specific CRISPR/Cas9 approach, cleaving the

mutant DNA at a SNP-derived PAM in the KRT12 gene in the mouse cornea

(Courtney et al. 2016). For this approach, we took advantage of the

discriminating nature of the PAM, the recognition site for the CAS enzyme,

which requires a precise sequence composition for the DSB to occur. When

a DNA mutation or single nucleotide polymorphism (SNP) is known to

cause disease also creates a PAM where none existed before, this mutation


Table 4. Corneal dystrophies: associated inheritance pattern, gene

locus and causative genes

Corneal

layer

Disease Inheritance

Pattern

Genetic

Locus

Gene

known

Gene/s

Affected

IC3D Category

Epithelial and

Sub-Epithelial

Dystrophies

EBMD Minority of

cases, mostly

sporadic

5q13 Some cases TGFBI Some C1

ERED Autosomal

Dominant

Unknown Unknown N/A C4

(Smolandiensis

variant = C3)

SMCD Autosomal

Dominant

Unknown Unknown Unknown C4

MECD Autosomal

Dominant

12q13 and

17q12

Yes KRT3 and

KRT12

(Stocker–

Holt variant)

C1

LECD X-chromosomal

dominant

Xp 22.3 Unknown Unknown C2

GDCD Autosomal

Recessive

1p32. Yes TACSTD2,

previously

M1S1

C1

Bowman

Layer

Dystrophies

RBCD Autosomal

Dominant

5q13 Yes TGFBI C1

TBCD Autosomal

Dominant

5q13 Yes TGFBI C1

10q24 Unknown Unknown C2

GWCD Autosomal

Dominant

Unknown Unknown Unknown C4

Stromal

Dystrophies

LCD Autosomal

Dominant

5q13 Yes TGFBI C1

Autosomal

Dominant

9q34 Yes GSN C1

GCD Autosomal

Dominant

5q31 Yes TGFBI C1

MCD Autosomal

Recessive

16q22 Yes CHST6 C1

SCD Autosomal

Dominant

1p36 Yes UBIAD1 C1

CSCD Autosomal

Dominant

12q21.33 Yes DCN C1

FCD Autosomal

Dominant

2q35 Yes PIP5K3 C1

PACD Autosomal

Dominant

12q21.33 Yes KERA,

LUM,DCN,

EPYC

C3

CCDF Unknown Unknown Unknown Unknown C4

PDCD Reported AD Unknown Unknown Unknown C4


Table 4. (Continued)

Corneal

layer

Disease Inheritance

Pattern

Genetic

Locus

Gene

known

Gene/s

Affected

IC3D Category

Descemet's

Membrane

and

Endotehlial

Dystrophies

FECD Unknown 13pTel –

13q12.13,

15q,

18q21.2 –

q21.32.

Unknown Unknown C3 or C4

Autosomal

Dominant

1p34.3 –

p32

Yes COL8A2 C1

PPCD Autosomal

Dominant

20p11.2–

q11.2

Unknown Unknown C2

1p34.3–

p32.3

Yes COL8A2 C1

10p11.2 Yes ZEB1 C1

CHED 1 Autosomal

Dominant

20p 11.2–

q11.2

(pericentrom

eric region)

Unknown Unknown C2

CHED2 Autosomal

recessive

20p13

(telomeric

portion)

Yes SLC4A11 C1

XECD X-chromosomal

dominant

Xq25. Unknown Unknown C2

Total 23 Known = 20 Known = 17 Known = 10

Partially

known = 4

Unknown =

9

can be exploited to precisely target the mutant allele with the CRISPR/Cas

system. In this study, a disease causing missense mutation, p.Leu132Pro

which generates a novel PAM site was identified for Meesmann's epithelial

corneal dystrophy (MECD) within the KRT12 gene (Courtney et al. 2016).

MECD most commonly occurs as an autosomal dominant genetic disease,

and we were able to knock down the mutant allele via NHEJ while leaving

the wild type allele intact. This approach of allele specific gene editing could

be used to treat other ocular dystrophies caused by dominant negative

missense mutations that generate novel PAMs, further expanding the

potential for CRISPR/Cas9-based therapeutics.


3.3. Mutant Allele Targeting, a Dual Cut Approach

There are at least 70 known missense mutations located in the coding region

of the TGFBI gene that can lead to various forms of corneal dystrophy. It is

neither possible nor realistic to design a sgRNA for each of these mutations,

and since at least 44 of the 70 mutations occur adjacent to a naturally

occurring PAM, stringent fidelity is required to achieve complete allele-

specificity to avoid the off target site that exists in the form of the wild-type

allele for these mutations (Christie et al. 2017). There have been studies

which demonstrated that truncating the sgRNA leads to an increased target

specificity and reduced off target activity (Pattanayak et al. 2013, Fu, Reyon,

and Joung 2014, Fu et al. 2014). However, it has been shown by our group

that a range of truncated guide lengths or addition of 5′-GG to the 20 nt

guide sequence did not provide a marked improvement of specificity for

a sub group of the TGFBI mutations (Figure 2) (Christie et al. 2017). We

therefore have conceived of a dual cut approach that in theory would achieve

knock down for all the TGFBI missense mutations applicable to

heterozygous genotypes (Christie KA 2019). With a dual cut approach, the

treatment of autosomal disease like TGFBI corneal dystrophy is not

constrained by the specific mutation responsible for any given phenotype.

Our approach utilises natural occurring variants in the target region

characterized by SNP-derived PAMs, which allow for the design of allele-

specific SNP-derived PAM sgRNAs (Christie KA 2019). Utilising natural

variants in the target region that harbour a PAM which also lie in cis with

the causative mutation allows this method to discriminate between the wild-

type and mutant alleles. Premature termination of translation via NHEJ

brings on permanent disruption of the target allele. A dual guide approach

to CRISPR/Cas9 editing was shown to be a promising therapeutic approach

for the treatment of patients with LCA10 bearing the CEP290 splice

mutation, (Ruan et al. 2017a) and in a human cell line it was demonstrated

that DNA deletions up to 10 kb coupled to precise NHEJ effectively knocked

down a mutant allele (Zheng et al. 2014). In order to generate a therapeutic

outcome for the TGFBI mutation targeted in our work, an in cis dual guide

approach was required (Christie KA 2019).

Figure 2. Guide-length screen to determine the effect on specificity of a guide-specific system. Reports have indicated that truncating the

length of the matching sequence within the guide from 20 to 18 nucleotides can reduce genome-wide off-target cutting, while

maintaining on-target efficiencies. An assessment of the effect of guide-length upon specificity between the wild-type and mutant

alleles, using a dual-luciferase assay, was conducted for the 5 most prevalent TGFBI mutations. Reports have shown that guide lengths

<16nt abolish cleavage activity. For each mutation a range of guide lengths from 16 to 22 nucleotides were tested, each guide was

targeted to the wild-type and respective mutant sequence and the firefly luciferase activity was measured as an indicator of specificity.

For all mutations investigated, the truncated guides did not provide a marked improvement of specificity; for most cases maximal

discrimination occurred with guides 20 or 19 nucleotides in length. For R124C, a 20nt guide seemed to confer allele-specificity,

however, no other guide length offered any adequate discrimination (Figure 2a). In the case of the R555Q mutation, guides in the 18–

20nt range did not offer sufficient discrimination, although, interestingly, the 21nt guide provided convincing allele-specificity (Figure

2d). R555W did not offer any considerable allele-specificity for any length tested (Figure 2e). R124H and R124L displayed clear allele-

specific cleavage, especially in the 18–20nt sgRNA range, with minimal cutting of the wild-type sequence (Figure 2b and c).

Interestingly for the R124 mutations guide lengths of 21nt seemed to impair cleavage activity in all cases.


While our study focussed on the issue of on-target allele-specificity in

relation to the TGFBI corneal dystrophies, for translation to the clinic a

number of key hurdles will need to be overcome, including genome wide

specificity and efficiency of delivery to the correct cells in the cornea. Potent

targeting of the correct cell population must be achieved. In corneal tissue,

the majority of TGFBI protein is produced in the epithelium, and the

epithelium is continually turned over and repopulated via the limbal

epithelial stem cells (LESCs). Thus, in order to permanently correct the

TGFBI corneal dystrophies efficient delivery to the LESCs must be achieved

(Christie KA 2019).

Another application for utilizing a dual cut CRISPR/Cas9 approach in

the context of the cornea involved a study aimed at understanding the

function of the PAX6 gene in maintaining the identity of human corneal

epithelial cells (CECs) (Kitazawa et al. 2017). PAX6 is a transcription factor

and is necessary for eye development. To investigate how PAX6 regulates

the gene expression profile of CECs, this study designed a two guided RNA

system targeting specific genomic loci to make a DSB that resulted in a

nonsense mutation upon NHEJ. This resulted in a complete knock down

PAX6 expression. The PAX6 knockout via the dual cut CRISPR/Cas9 system

resulted in a loss of CEC identity through the downregulation of corneal

epithelial-related genes and the upregulation of epidermis and

keratinization-related genes. Specifically, PAX6-depleted CECs resulted in

down-regulated genes that were primarily CEC-specific and included

keratin 12, keratin 3, clusterin (CLU), aldehyde dehydrogenase 3 family

member A1 (ALDH3A1), angiopoietin-like 7 (ANGPTL7) and transketolase

(TKT), while up-regulated genes were primarily epidermis-related and

included keratin 10, keratin 1, involucrin (IVL) and filaggrin (FLG). The

findings suggested that PAX6 maintains CEC identity by regulating

differentiation (Kitazawa et al. 2017).


3.4. Multiplexing in the CRISPR System for Multigene

Ocular Disease

Complex genetic ocular diseases like primary open angle glaucoma

(POAG) or AMD where dysregulation at multiple loci contribute to the

overall disease phenotype could be treated by multiplexing the CRISPR

system. Likewise, retinal dystrophies are genetically heterogeneous diseases

(Berger, Kloeckener-Gruissem, and Neidhardt 2010, Roosing et al. 2014,

Zeitz, Robson, and Audo 2015). An advantage of the CRISPR/Cas system

over protein recognition-based methods is its ability to perform multiplex

genome editing at high efficiencies by expressing or providing multiple

guide RNAs (Cong et al. 2013, Mali, Yang, et al. 2013). Table 5 describes

current approaches for providing multiple gRNAs in vivo. More recently,

multiplexing was achieved with AAV-delivered Cas9 nuclease. A

quadruplex gRNAs/SaCas9 vector consisting of SaCas9 and multiplex

sgRNAs was successfully delivered using AAV-DJ/8 for in vivo excision of

HIV-1 proviral DNA in various solid tissues/organs via a single intravenous

injection in humanized bone marrow/liver/thymus (BLT) mice with chronic

HIV-1 infection (Yin, Zhang, et al. 2017).

A construct referred to as CRISPR-on was used in a study where an

exogenous reporter gene in mouse and human cells was activated enabling

the simultaneous activation of multiple endogenous genes (Cheng et al.

2013). The system utilized a nuclease-dead Cas9 (dCas9) protein fused with

a transcriptional activation domain and a single guide RNAs (sgRNAs) with

complementary sequence to gene promoters. The ability a system like

CRIRPR-on to regulate endogenous genes is valuable for the study of gene

functions and has great potential for therapeutic applications.

Another study found that by introducing different Cas9 proteins they

could mediate DNA cleavage or regulate transcriptional activity

simultaneously (Esvelt et al. 2013). Catalytically active Cas9 and distinct

sgRNA constructed with 14-15-bp target sequences and MS2 binding loops

activated gene expression without inducing DSBs. Co-expression of full

Table 5. Current approaches for providing multiple gRNAs

Multiplex CRISPR System Description

Multigene cassettes to express several

gRNAs

A lentiviral system was used to express a Cas9 variant, a reporter gene and up to four sgRNAs from

independent RNA polymerase III promoters incorporated in the vector with the Golden Gate cloning method.

Each sgRNA was efficiently expressed and was able to mediate multiplex gene editing (Kabadi et al. 2014).

The expression cassettes of gRNAs were tandemly ligated into a single vector using the Golden Gate cloning

method. Additionally, Cas9 nickase-mediated genome editing was constructed with a vector expressing Cas9

nickase and six gRNAs targeting three genomic loci (Sakuma, Sakamoto, and Yamamoto 2017).

Guide RNAs post-transcriptionally

processed from a single transcript using

Csy4-based excision

An RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) tool utilizing multiple RNA

regulatory strategies, including RNA-triple-helix structures, introns, microRNAs, and ribozymes, with Cas9-

based CRISPR-TFs and Cas6/Csy4-based RNA processing gives the ability to modulate endogenous

promoters including multistage cascades and RNA-dependent networks (Nissim et al. 2014).

tRNA-dependent cleavage of gRNAs A general strategy to produce numerous gRNAs from a single polycistronic gene was utilized to boost the

targeting and multiplex editing capability of a CRISPR/Cas9 system. The study demonstrated that synthetic

genes with a tandemly arrayed tRNA–gRNA architecture, efficiently and precisely processed gRNAs to

desired 5′ targeting sequences in vivo and directed Cas9 to edit multiple chromosomal targets (Xie,

Minkenberg, and Yang 2015).

Arrays to express multiple crRNAs for Cpf1 Using an artificial CRISPR pre-crRNA array consisting of four spacers separated by direct repeats (DRs) from

the CRISPR locus of Francisella novicida (FnCpf1) and two Cpf1 orthologs with activity in mammalian cells,

Acidaminococcus Cpf1 (AsCpf1) and Lachnospiraceae Cpf1 (LbCpf1) multiplex gene editing was observed

(Zetsche et al. 2017).

Self-cleaving ribozyme flanked gRNAs Multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a

single U6 promoter to exert genome editing. The study provided a novel strategy to enable tissue-specific

expression of more than one gRNAs for multiplex gene editing (Xu et al. 2017).

Direct introduction of Cas9 proteins

preloaded with different gRNAs Cas9 RNPs

A method for the rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein

complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or

electroporation was described. This approach for multigene targeting in Jurkat cells resulted in two-locus and

three-locus indels in approximately 93% and 65% of the resulting isolated cell lines with a reduced off-target

cleavage rate (Liang et al. 2015).


length normal sgRNA and truncated sgRNA fused with MS2 binding loops

can simultaneously cleave or activate transcription at the respective target

genes (Dahlman et al. 2015).

4. DELIVERY OF CRISPR/CAS TO THE EYE -

IN VIVO AND EX VIVO

Efficient and effective delivery of the CRISPR/Cas machinery to the

tissues or cells of interest is one of the most important aspects for translating

the therapy to the clinic. The physiology of the eye presents challenges for

the pharmacologist and formulation scientist for the design of efficient drug

delivery systems. Delivery of drugs to the targeted ocular tissues is restricted

by various precorneal, dynamic and static ocular barriers (Patel et al. 2013).

Figure 3. Delivery of Cas9-sgRNA complex. A. Viral vector based delivery B.

Physical method of microinjection based delivery C. Nanoparticle based delivery.

The outermost surface of the eye has many barriers that prevent entry of a

foreign substance. Biological barriers must be considered for any

CRISPR/Cas system to be delivered to the eye. Many avenues of delivery

have been explored, broadly sub-divided into viral and non-viral approaches


(Figure 3). An ideal vehicle for gene therapy should have a large capacity to

package all the CRISPR/Cas9 machinery, cell tropism for the specific cell

population of interest, high nucleus targeting efficiency along with minimal

immune response and cytotoxicity.

4.1. In Vivo Systems

A range of viral systems including adenoviruses, AAVs and

retroviruses, specifically lentivirus, has been investigated for their potential

to deliver genetic payloads to transduce various types of tissues, including

the eye (Kay 2011). Adenovirus has the advantage of a large packaging

capacity but initiates a substantial immune response; (Borras et al. 2001,

Tsubota et al. 1998) whereas, AAV provides efficient transduction with a

minimal immune response and persistence in the nucleus but has a limiting

packaging capability (Sharma et al. 2010, Zinn and Vandenberghe 2014).

Lentivirus has a considerable packaging capacity but can cause unwanted

integration (Athanasopoulos, Munye, and Yanez-Munoz 2017). Non-

integrating viruses like adenoviruses transiently express the transgene

making them ideal for applications with CRISPR/Cas system, as limited

Cas9 expression reduces the off target cleavages.

AAV has been the leading vector of choice for gene therapy, particularly

ocular gene therapy. Recently AAV technology has been deployed for

delivering CRISPR/Cas9 components to investigate gene function and

disease treatment in the mammalian eye (Huang et al. 2017, Ruan et al.

2017b, Yu et al. 2017, Kim, Koo, et al. 2017). AAV has been shown to

transfer genes successfully into photoreceptor cells and retinal pigment

epithelium (RPE) of the mouse retina with low cytotoxicity (Day et al.

2014). Sub-retinal injection of an AAV vector has been shown to be safe and

effective in animal studies, (Acland et al. 2001, Pang et al. 2006) and human

trials (Boye et al. 2013, Cideciyan et al. 2009, Jacobson et al. 2012,

MacLaren et al. 2014, Testa et al. 2013, Trapani et al. 2015, Pierce and

Bennett 2015). The use of AAV-2 based gene augmentation therapy for

RPE65-associated retinal degeneration gene therapeutics has been


extensively tested in clinical trials (Pierce and Bennett 2015). However,

AAV-2 was found to be not as effective as other AAV serotypes for

transduction within the cornea, and as such a variety of other AAV vectors

have been shown to successfully transduce the cornea with varying success

(Sharma et al. 2010, Liu et al. 2008). A study reported that a hybrid serotype,

AAV-2/8, delivered by an intrastromal injection was able to transduce long

term expression of the transgene in the stromal keratocytes in both ex vivo

human and in vivo mouse models (Hippert et al. 2012). An intracameral

injection of the AAV hybrid serotype vector, AAV-2/9 was shown to

transduce corneal endothelial cells in mouse (O'Callaghan et al. 2017). In

our hands AAV- 2/9 was effective in transducing both the corneal epithelium

and corneal endothelium cells (Unpublished Data). Lastly, a novel synthetic

AAV has been reported to efficiently transduce corneal stroma and

endothelial cells following intrastromal injection in mouse (Wang et al.

2017).

With AAV vectors being the leading choice of gene therapy in

ophthalmology, different strategies have been applied to circumvent their

limiting packaging capacity. A dual AAV vector system was utilized to

deliver the the CRISPR components, the sgRNA and SpCas9 in two separate

AAV vectors and delivered in vivo to post mitotic photoreceptor cells in

experiments utilizing retinal degeneration mouse models (Yu et al. 2017).

Since the crystal structure of at least two Cas9 proteins complexed with

guide RNA and target DNA have been determined, (Nishimasu et al. 2014,

Yamano et al. 2016) the way has been paved for designing modifications to

the complex which could be used to overcome the payload restrictions of the

AAV vectors. Also, to limit an AAV mediated gene therapy’s persistent

transgene expression, the use of tissue-specific promoters or self-cleaving

vectors have been investigated. A tissue-specific reporter ensures expression

is localised to only the desired target tissue. In 2 clinical trials where LCA

was treated with gene replacement of RPE65, an AAV-2 vector with a

human RPE65 promoter, hRPE65 was used to minimise unwanted

expression in other tissues (Bainbridge et al. 2008). With respect to gene

editing with a CRISPR system, minimising expression of Cas9 may help to

reduce off-target effects (Moore et al. 2018). The use of a self-cleaving


system which involves introducing both a sgRNA targeted to the gene of

interest and a sgRNA targeted to the delivery vector could also be used to

minimise the period of transgene expression and off-target effects (Chen et

al. 2017, Moore et al. 2014, Petris et al. 2017).

As an alternative to non-viral delivery systems, broadly classified into

two sub categories are physical and chemical systems (Ramamoorth and

Narvekar 2015). Unlike viral vectors, non-viral systems are not limited by

packaging restrictions, and they have lower immunogenicity and toxicity

(Pack et al. 2005, Mintzer and Simanek 2008). Physical methods including

injection of naked DNA, electroporation, gene gun, ultrasound and

magnetofection utilise force to increase cell permeability (Mohan et al.

2005, Oliveira, Rosa da Costa, and Silva 2017). Chemical delivery methods

employ the principal of electrostatic interactions between the negatively

charged nucleic acid and either a natural or induced positive charge of the

carrier, generating a complex with an overall positive charge (Jin et al.

2014). Through carrier design alteration and by targeting the ligand

decoration on the cell types, non-viral vectors can deliver therapeutic nucleic

acids to target tissues or cells. Several naturally occurring and synthetic

cationic polymers have been used for the delivery of nucleic acids. A novel

hyaluronic acid-chitosan nanoparticle was used to transfect efficiently the

corneal and conjunctival cells in rabbits in vivo and in a human in vitro

model, (de la Fuente, Seijo, and Alonso 2008a, b) and ultrapure chitosan

oligomers were used as carriers for transferring a luciferase plasmid to

keratocytes in rat cornea (Klausner et al. 2010). Interestingly, another report

developed an artificial virus using a nanoparticle, liposome-protamine-DNA

complex (LPD), modified with a cell permeable peptide and a nuclear

localization signaling (NLS) peptide, to deliver a functional Rpe65 gene to

mouse eyes improving vision in vivo (Rajala et al. 2014). Thus, LPD

nanoparticles provide a promising, efficient, non-viral method of gene

delivery with clinical applications.

Other promising delivery systems utilising the advantages of non-viral

delivery are CRISPR/Cas9 RNP complexes (Zuris et al. 2015) and CRISPR-

Gold which is able to simultaneously deliver RNPs and donor DNA into

cells resulting in successful in vivo HDR (Lee et al. 2017). A recent work


demonstrated that a structure-guided chemical modification of guide RNA

delivered using a non-viral lipid nanoparticle resulted in a >80% gene

editing of Pcsk9 (Yin, Song, et al. 2017). The advantage of using a direct

protein-packaged approach is that components of the CRISPR complex have

a short half-life and are not being continuously synthesized in the cell once

the genomic modification has occurred. As no delivery system on its own

meets all requirements, therapeutic genome editing could be best achieved

by combining a viral coupled to non-viral delivery. For example, Cas9

mRNA delivered using lipid nanoparticles along with AAV encoding a

sgRNA and a repair template has been shown to induce repair of Fah in a

mouse model (Yin et al. 2016). These studies pave the way for more efficient

use of alternative in vivo delivery systems for gene editing especially in the

eye.

4.2. Ex Vivo Systems

Stem cell therapies for the treatment of AMD and other degenerative

retinal disorders including inherited retinal disease are positioned to make

an impact on clinical care (Wood et al. 2019). Stem-cell-based photoreceptor

cell replacement has been investigated in different cell types and in animal

models. The CRISPR/Cas9 system was successfully employed in embryonic

stem cells (ESCs) where efficient gene correction was demonstrated in a

mouse cataract model (Wu et al. 2013). A study utilizing human embryonic

stem cells (hESCs) demonstrated the ability of a hESC-derived retina to

form structured mature photoreceptor layers after transplantation into nude

rats and two different monkey models (Shirai et al. 2016). Human retinal

progenitor cells (hRPCs) from fetal neural retina were transplanted into a rat

model where at 12 weeks after treatment, hRPC-grafted eyes had

significantly superior visual acuity compared to non-treated eyes (Luo et al.

2014). For the study of optic nerve biology and disease, a CRISPR

engineered reporter cell line was able to demonstrate the differentiation of

hESCs to retinal ganglion cells (RGCs) (Sluch et al. 2015). Although hESCs

offer extensive differentiation capabilities allowing for the study of very


early stage development of life as well as providing a source of cells for the

development of novel therapeutic molecules and cell-based therapies, their

use is controversial. Due to the ethical and practical reasons of using cells

derived from embryonic or fetal tissue, much work and effort has been

conducted with patient-derived induced pluripotent stem cells (iPSCs),

which offer the best possible immunologic match to the patient (Burnight et

al. 2017).

The use of iPSCs in gene editing technologies hold great promise in the

treatment of inherited disease. CRISPR/Cas tools enable precise

manipulation of these stem cells for ophthalmic applications providing a

platform for correction of deleterious mutations in a patient's own cells. For

three different types of variants that lead to retinal diseases, CRISPR/Cas9

mediated genome editing strategies were used to target and correct the

mutations in patient-derived iPSCs. A HDR strategy targeting an Alu

insertion in the MAK gene was able to restore the retinal transcript and

protein; the CRISPR-Cas9-mediated NHEJ approach was able to excise the

IVS26 cryptic-splice mutation in CEP290 demonstrating a correction of the

transcript and protein in patient iPSCs; and lastly, with allele-specific

CRISPR guides, the Pro23His rhodopsin (RHO) allele was selectively

targeted which, following delivery to both patient iPSCs in vitro and pig

retina in vivo, created a frameshift and premature stop preventing

transcription of the disease-causing variant (Burnight et al. 2017).

Additionally, in patient-specific iPSCs an RPGR point mutation that causes

X-linked RP was precisely repaired (Bassuk et al. 2016).

While iPSC driven ex vivo therapy for inherited corneal disease has not

yet been realized, the potential and the development of cell-based therapies

using stem cells represent a significant breakthrough in treatment options in

regenerative medicine for the ocular surface. There have been several studies

focused on differentiating iPSCs into corneal epithelial cells for the

treatment of limbal stem cell deficiency (LSCD) (Saghizadeh et al. 2017).

One such study was able to successfully induce corneal epithelial cells from

both human adult corneal limbal epithelial cells (HLEC)-derived iPS cells

and human adult dermal fibroblast (HDF)-derived iPS cells by the stromal

cell-derived inducing activity (SDIA) differentiation method (Hayashi et al.


2012). Another study was able to derive iPSCs from human primary LSCs

and re-differentiate these iPSCs back into the limbal corneal epithelium

(Sareen et al. 2014). Our group previously reported the ability to culture

LESCs from a patient with MECD. The causative mutation, p.Leu132Pro in

the KRT12 gene in these cultures was effectively silenced using siRNA

(Courtney, Atkinson, Allen, Moore, Walsh, Pedrioli, MacEwen, Pellegrini,

Maurizi, and Serafini 2014).

On-going research with corneal cell models has advanced the potential

for the CRISPR/Cas9 complex to be delivered ex vivo for the treatment of

inherited corneal disease like the TGFBI corneal dystrophies. Efficient

corneal transduction resulting in transgene expression via intra-stromal

injection of an AAV2/8 vector was successful in human corneal explants

(Hippert et al. 2012) An AAV8/9 chimeric capsid was also shown to

transduce human corneal explants in a gene replacement study for the

treatment of cornea clouding brought on by mucopolysaccharidosis type 1

(MPS1) (Vance et al. 2016). While an AAV approach to cellular

transduction has shown promise, there are alternative methods to AAV and

viral transduction. The variant within the TGFBI gene that causes granular

corneal dystrophy type 2 (GCD2) was repaired in human corneal keratocytes

via a CRISPR/Cas9-induced HDR mechanism (Taketani et al. 2017). In this

work a plasmid vector, px458 which enabled bicistronic expression of

SpCas9 and green fluorescence protein (GFP) was co-transfected into

primary GCD2 mutant human corneal keratocytes with a single stranded 100

base oligonucleotide (ODN) that served as donor repair template. This study

was the first to demonstrate in vitro gene correction in mutant human

primary corneal cells using CRISPR/Cas9 and HDR (Taketani et al. 2017).

The importance of an ex vivo approach to the delivery of therapeutic

cells containing the CRISPR/Cas9 complex (Figure 4) for the treatment of

autosomal dominant disease such as GCD2 and other TGFBI corneal

dystrophies is crucial when NHEJ resulting in a knock down of the mutant

allele is not a viable option. We have established that the TGFBI protein is

necessary for the wound healing process (Christie KA 2019). As such,

individuals who carry homozygous mutations for an autosomal dominant

TGFBI corneal dystrophy cannot be treated with allele specific


CRISPR/Cas9 NHEJ gene editing since, with this method both alleles

expressing the TGFBI transcript would be silenced. HDR within a corneal

stem cell model could potentially be used to replace mutant corneal cells

within an individual who carries the mutation in both copies of the TGFBI

gene. However, ex vivo stem cell transplantation of gene edited corrected

cells via HDR is dependent on removing all resident mutations carrying

LESCs, and for those TGFBI mutations resulting in an intrastromal

pathology, stem cell transplantation will not undo existing stromal

pathology. As such, a corneal dystrophy such as MECD which affects the

anterior corneal epithelium (McLean and Moore 2011) is well suited for ex

vivo stem cell replacement. This approach would be most advantageous for

targeting the MECD SNP-derived PAM mutation, p.Leu132Pro in the

KRT12 gene (Courtney et al. 2016).

Figure 4. CRISPR therapeutic approach; transition to clinics.

5. ANIMAL MODELS FOR EYE DISEASE

The generation of new model systems with genome-editing tools, in

particular those based on CRISPR/Cas9, are opening new avenues for both

research into, and for, the treatment of eye disease. Genome-editing

technologies are allowing researchers to take full advantage of both animal

and cellular models, and to work more easily with non-traditional model

organisms for eye research. In ophthalmology, these animal models are


important first steps for examining genetic conditions as well as assessing

therapeutics for such genetic diseases. For generating a transgenic animal

model, the CRISPR/Cas9 system has multiple advantages over the ZFN or

TALEN approach to gene editing. The gRNA determines the specificity of

CRISPR systems and not the nuclease; therefore, the CAS protein can

remain unchanged between target sites and between different organisms.

Adjusting the target with a change to the gRNA sequence allows for higher

efficiencies when compared to systems that require protein alterations or

changes to embryonic stem cells (ES). Since homologous recombination is

generally required to insure a site‐specific edit within ES cells, and given

that a DSB can increase the probability of homologous recombination,

CRISPR/Cas9 increases specificity and site‐specific genome targeting

frequency in view of the system’s effectiveness in producing DSBs (Zarei et

al. 2019). The emergence of this technology has allowed the genome editing

in mouse embryos to be quicker and more cost-efficient (Yang et al. 2013).

In addition, CRISPR/Cas9 has revolutionized the generation of a wide

variety of transgenic animals such as zebrafish, (Ablain et al. 2015) rats,

(Bakondi et al. 2016) monkeys, (Zuo et al. 2017) pigs, (Yan et al. 2018) and

rabbits (Yuan et al. 2016).

Zebrafish represent a powerful model for studying vertebrate eye

development. Easy access to eye tissue for imaging and drug/gene delivery,

short lifecycle, and genetic similarities to higher vertebrates and mammals

gives zebrafish many advantages over other model organisms to explore the

applications of CRISPR/Cas gene editing in the study of important pathways

and regulators in the development of ocular tissue (Richardson et al. 2017).

Several studies demonstrate the power of CRISPR/Cas genome editing in

zebrafish. CRISPR/Cas gene editing was applied in the zebra fish model to

understand the role of membrane frizzled related protein (MFRP) in the

development of nanopthalmic-hyperopia; (Collery et al. 2016) SpCas9-

generated knockouts and morpholinos to show the importance of NeuroD in

photoceptor regeneration; (Taylor et al. 2015) and to show retina

regeneration and Müller glia de-differentiation (Yin et al. 2015, Serifi et al.

2016). In another non-mammalian animal model, an amphibian, Xenopus

tropicalis was used to create a highly penetrant and rapid retinoblastoma


(Rb) model. CRISPR/Cas9 gRNAs were injected into X. tropicalis embryos

for rapid identification of targets for therapeutic intervention (Naert et al.

2016). These studies demonstrate the important role of CRISPR/Cas genome

editing in animal models like the zebrafish for investigating diseases of the

human eye.

CRISPR/Cas systems are also applied to mammalian models of the eye.

The following examples focus on research conducted with mouse models

for the treatment of different eye diseases. In a study of a particular LCA

model, CRISPR-engineered mosaicism reveals that the loss of Kcnj13

function in mice mimics the human disease phenotypes (Zhong et al. 2015).

RP is a challenging retinal dystrophy for ophthalmologists. A mouse model

mimicking clinical phenotypes of RP was generated using CRISPR like

knock-in with the p.Leu135Pro, an RP-associated variant (Arno et al. 2016).

The most commonly studied preclinical model of RP called the “rodless”

(rd1) mouse is homozygous for two mutations, a nonsense point mutation

(Y347X) and an intronic insertion of a leukemia virus (Xmv-28) (Wu et al.

2016). Utilizing a CRISPR/Cas9 system, it was demonstrated that the

Y347X mutation is the causative variant in this form of RP. This group was

the first to demonstrate homology-directed recombination–mediated gene

correction in the visual system using the CRISPR/Cas9 system (Wu et al.

2016). Our research on MECD utilized a humanized mouse containing a

mutant KRT12 allele mutation, p.Leu132Pro (Courtney et al. 2016).

Through CRISPR/Cas9 directed NHEJ repair resulting in frame-shifting

deletions within the mutant KRT12 allele, we were able to demonstrate

reduction of the mutant KRT12 mRNA expression. The CRISPR/Cas9

system has also been successfully employed in stem cell lines where

efficient gene correction was demonstrated in a mouse cataract model (Wu

et al. 2013).

Pre-clinical studies in other CRISP/Cas9 edited mammalian models

include rabbits and pigs. A group studying GJA8, a gene known to be

involved in maintaining lens opacity and proper lens development, created

a gene knockout via a co-injection of Cas9/sgRNA mRNA into rabbit

zygotes. Their results showed that gene mutation efficiency in the GJA8

locus reached 98.7% in the rabbit embryos and that impaired GJA8 function


caused microphthalmia, small lens size and cataracts; knocking out GJA8 in

rabbit via CRISPR/Cas9 system caused human-like cataracts (Yuan et al.

2016). In research involving neurodegenerative Huntington’s disease (HD),

a large CAG repeat (150 CAGs) via somatic cell nuclear transfer (SCNT) in

combination with CRISPR/Cas9, was inserted into the endogenous pig HTT

gene generating a HD knockin (KI) pig model that expressed full-length

mutant HTT at the endogenous level (Yan et al. 2018). HD results from this

monogenetic CAG repeat expansion located in exon1 of the HTT gene.

There are advantages to using large mammals such as pigs over rodent and

rabbit models. Pigs are genetically, anatomically, and physiologically closer

to humans, and their fast breeding period and large litter size hold

advantages over non-human primates when considering the time line of

generating large animal models of human diseases (Yan et al. 2018).

6. RECENT PROGRESS IN CLINICAL GENE THERAPY

TRIALS FOR OCULAR DISEASES

Research for transitioning effective gene therapy strategies for ocular

disease to the clinic has rapidly grown in the past 15 years, particularly for

the treatment of retinal diseases. Clinical trials involving retinal gene therapy

are creating hope for future therapies for afflicted patients. They all employ

gene augmentation for loss of functions diseases, rather than gene editing

strategies. The most successful example of ocular gene therapy is by gene

replacement using AAV delivery (Pierce and Bennett 2015). It has been used

successfully for RPE65, Leber's congenital amaurosis 2 (LCA2), an early

onset form of autosomal recessive retinal degeneration caused by mutations

in the RPE65 (RPE-specific 65 kDa protein) gene. Ground breaking trials

utilising AAV vectors to deliver RPE65 did show safety and initial vision

improvements (Bennett et al. 2016, Jacobson et al. 2015). These studies were

the first to demonstrate proof of concept for gene therapy in human retinal

disease. Three separate phase I–II clinical trials were initiated, which yielded


promising results after subretinal administration of AAV2-hRPE65 vectors

(Bainbridge et al. 2008, Hauswirth et al. 2008, Weleber et al. 2016).

The three studies differed in terms of dose, inclusion criteria, type of

promoter, location of the injection and outcome measures. All the studies

reported high levels of safety and demonstrated efficacy up until the 3-year

follow-up. In 2018 subsequent results were reported for the trial involving

the AAV vector expressing RPE65 (rAAV2-CB-hRPE65) (Weleber et al.

2016, Pennesi et al. 2018). The outcome after 5 years was found to be small

and not sustained; however, there were no clinically significant adverse

events. The group concluded that treating patients at a younger age is

associated with better visual function outcomes during 5 years after

treatment (Pennesi et al. 2018). In a follow up study for the trial

administering the rAAV2/2 RPE65 vector, (Bainbridge et al. 2008) results

indicated that the amount of rAAV2/2 RPE65 required to drive the visual

cycle in affected persons was not met to the extent required for a durable,

robust effect (Bainbridge et al. 2015). Gene therapy for LCA is the first

ocular gene therapy to complete a phase III clinical trial for patients with

confirmed biallelic RPE65 mutation-associated retinal dystrophy (Jiang, Xu,

and Tsang 2018).

Another gene therapy presently at the pre-clinical trial stage, targets

wAMD with an AAV vector encoding aflibercept (ADVM-022) (Grishanin

et al. 2019). Vascular endothelial growth factor (VEGFA) plays a key role

in the development of wAMD, and as such VEGFA is targeted in the

treatment of the disease. Administering recombinant anti-VEGFA proteins

such as aflibercept with intravitreal (IVT) injections every 4 to 8 weeks is

an approved therapy for this disease. Aflibercept is a recombinant chimeric

protein consisting of the VEGFA binding portion of human VEGFR-1 and

VEGFR-2 fused to the Fc portion of human IgG1 immunoglobulin (Ashraf

and Souka 2017). ADVM-022 utilizes a variant of the AAV2 capsid as a

vector for delivering and encoding aflibercpt. It was reported that the

administration of ADVM-022 in non-human primates was tolerated with no

serious adverse safety-related findings and that more than one year past a

Table 6. Current clinical trials of gene therapy in eye

Disease Phase of clinical trial Delivery Gene therapy strategy Study plan and results

Leber's congenital

amaurosis 2 (LCA2)

Multiple Phase I/II trials for

RPE65-associated LCA have

been either completed or are

ongoing.

Sub retinal AAV2-hRPE65 vector

carrying RPE65 gene

Patients in all trials exhibited several aspects of visual

improvements within a few months after treatment,

though with varying degrees (Bainbridge et al. 2008,

Bennett et al. 2016, Weleber et al. 2016, Hauswirth et

al. 2008).

Phase III clinical trial Sub retinal SPK-RPE65 An on-going study.(Jacobson et al. 2015)

Stargardt (Sanofi) 48 week Phase I/IIA dose-

escalation trial

Sub retinal injection Lentiviral vector

SAR422459, carrying the

photoreceptor specific

ABCA4 gene.

No significant changes in visual acuity in either the

treated or untreated fellow eyes for the enrolled 23

patients. To continue enrollment in the cohort of

youngest patients with early-onset Stargardt disease and evidence of rapid progression of disease (ages 6 to

26 years; all other cohorts involve patients 18 years or

older) (Parker et al. 2016).

MERTK-RP Phase I study Sub retinal AAV2 vector with an

RPE-specific promoter

driving MERTK

Study recently completed in Saudi Arabia. 6 patients

were treated without any serious adverse events.

Three of these patients displayed measurably

improved visual acuity in the treated eye following

surgery, although two of them had lost that improvement by two years (Ghazi et al. 2016).

Usher1B 48 week Phase I/IIA dose-escalation trial

Sub retinal Lentiviral vector SAR421869 (UshStat),

carrying Myosin 7A gene

Out of the 9 adult patients treated, a majority have shown an initial postoperative drop in BCVA and

visual fields that improved to baseline within two

weeks. The vision was stable (in either the treated or

untreated fellow eyes) after 48 weeks in a majority of

patients. (Unpublished Data).

Table 6. (Continued)

Disease Phase of clinical trial Delivery Gene therapy strategy Study plan and results

X-linked Retinoschisis

(XLRS)

48 week Phase I/IIA safety and

efficacy trial

Intravitreal AAV8-RS1 vector

carrying the gene

retinoschisin 1(RS1)

A study evaluating three different increasing dose

levels of an AAV8-RS1 vector in 9 participants with

pathogenic RS1 mutations with VA of 20/63 or worse

in one eye (NCT02317887) (Cukras et al. 2018). The second study is evaluating an AAV-RS1 vector in

up to 27 patients. It involves three initial groups of

adult patients receiving increasing dose levels of the

vector and will also evaluate the maximum tolerated

dose level in patients 6 years and older

(NCT02416622).

Choroideremia Multiple Phase I and II trials of

the AAV.REP1 vector are

ongoing at several sites

Sub retinal AAV-REP1 vector

carrying RAB escort

protein 1 gene

In all patients, the increase in retinal sensitivity over

six months in the treated eyes correlated with the

vector dose administered per area of surviving retina.

(Cideciyan et al. 2009). The early improvement

observed in two of the six patients was sustained at

3.5 years after treatment despite progressive

degeneration in the control eyes. (Edwards et al.

2016). Other trials of subretinal placement of the AAV.REP1 vector are ongoing, including a Phase I/II

trial (NCT02341807).

Achromatopsia Phase I/II dose-escalation study Sub retinal AAV-CNGB3 vector

carrying CNGB3 gene

The study is going on in patients with CNGB3

achromatopsia at four sites in the United States

(NCT02599922).

Age-related Macular

Degeneration (AMD)

Phase 1 study Intravitreal AAV2-sFLT01 The treatment was safe with no dose-limiting toxicity

(Heier et al. 2017).

Leber Hereditary Optic

Neuropathy (LHON)

Two Phase 3 trials, in Europe

and the U.S evaluating the

effectiveness of GS010 in early

onset LHON patients

Intravitreal rAAV2/2-ND4 (GS010) A study for patients with vision loss of six months or

less (NCT02652767), and another study is assessing

patients whose vision loss began more than six

months but less than a year ago (NCT02652780).


single IVT injection, ADVM-022 continued to provide robust aflibercept

expression. The effects of the ADVM-022 treatment were found to be

similar to a bolus treatment of aflibercept protein (Grishanin et al. 2019). A

single dose injection has clear advantages over a frequent dosing schedule

where lack of patient compliance may result in recurrence of active wAMD.

Gene-based therapy for wAMD has the potential to overcome such

compliance issues by ideally providing a life-long supply of the anti-

VEGFA protein following a single vector administration.

The remarkable success of the RPE65 and ADVM-022 studies has

paved the way for other clinical trials, particularly using AAV-mediated

gene delivery; however, there are challenges in delivering an effective and

enduring dose to stop cell death or degeneration. Active clinical gene

replacement trials are underway targeting other retinal diseases such as

Stargardt's disease, RP, Usher syndrome, X-linked retinoschisis (XLRS),

choroideremia, achromatopsia, AMD and Leber hereditary optic neuropathy

(LHON) (Table 6). These trials use either AAV or lentivirus as viral vectors.

7. CRISPR/CAS GENE EDITING TRANSITION TO CLINICS

CRISPR/Cas9 gene editing has undoubtedly shown great promise for

applications in gene correction therapy. The most imminent reason for

transitioning CRISPR/Cas research to clinics is direct therapeutic gene

editing in human cells, an advantage over viral-mediated gene therapy.

While no ocular gene therapy trials using CRISPR are yet underway, a

CRISPR gene editing technique has been approved by the National Institutes

of Health, US (NIH) to help extend cancer therapies that rely on enlisting a

patient’s T cells. Early in 2018 doctors at the University of Pennsylvania

were in the final steps of preparing for a clinical trial, and in 2019 two cancer

patients who are enrolled in this study were treated (Dearment 2019). This

trial is small and designed to investigate whether CRISPR is safe for use in

people. In the study, doctors will remove people’s blood cells, modify them

with CRISPR in the lab, and then infuse them back into the patients. The

study involves three different types of cancer: multiple myeloma, sarcoma,


and melanoma and proposes to remove T cells from at least 18 patients. Led

by Dr. Edward Stadtmauer, the protocol reprograms a person’s immune cells

to find and attack tumors. University of Pennsylvania scientists intend to use

CRISPR to edit two genes in patients’ T cells to make them better cancer

fighters. One of the genes to be edited, PD-1 makes a molecule that cancer

cells utilize to block the immune system from fighting the cancer. An

additional edit removes a natural T-cell protein that could interfere with this

process and in its place, an engineered receptor will steer the immune cells

toward particular tumors (Baylis and McLeod 2017) (Figure 4).

Similar studies have been under way in China where another trial is

making the first-ever attempt to use CRISPR to edit cells in the human body

infected with the human papillomavirus (HPV) while leaving the DNA of

normal cells untouched. (Figure 4). CRISPR would target and destroy the

viral genes of HPV that cause tumour growth. This study is to begin at the

First Affiliated Hospital of Sun Yat-Sen University in China (Le Page 2017).

In addition to this study, a research team in China edited the embryos of twin

girls born in November, 2018. In a highly controversial undertaking, a team

at the Southern University of Science and Technology in Shenzhen

purportedly eliminated the gene CCR5 via CRISPR editing in the hopes of

rendering the offspring resistant to HIV, smallpox, and cholera. If proven

successful, this effort would mark a stunning medical achievement;

however, this study and any future studies of this kind are ethically charged

because changes to an embryo could eventually affect the entire gene pool

if future generations were to inherit the edited gene. It goes without saying,

researchers and medical practitioners should adhere to ethical guidelines

such as those put forward by the Academy of Sciences in the US. Currently,

the use of genetically engineered embryos to establish a pregnancy is

prohibited in much of Europe, the United States and China (Regalado 2018).

The use of CRISPR is also under development for treating ocular

diseases. Editas Medicine and Allergan Pharmaceuticals International

Limited (Allergan) plan to initiate patient screening for clinical trials to test

the efficacy of EDIT-101, a CRISPR genome editing drug for the treatment

of Leber congenital amaurosis 10 (LCA10). They plan to enroll 10-20

patients in the U.S. and Europe. Utilizing an AAV5 viral vector that encodes


for the Staphylococcus aureus Cas9 along with two different gRNAs

targeted to either side of the IVS26 mutation between exon 26 and exon 27,

EDIT-101 is hypothesised to delete or invert the mutation such that

functional CEP290 expression is restored. Recently published results detail

the development of EDIT-101 and summarizes in vitro experiments in

human cells and retinal explants (Maeder et al. 2019). Subretinal delivery of

EDIT-101 was well tolerated and achieved sustained CEP290 editing in

photoreceptor cells in mice and non-human primates, which met or exceeded

the target therapeutic level. This study also determined that the 2 gRNAs

chosen for this treatment were highly specific, and no off-target sites could

be verified in either multiple human cell lines or human retinal cells.

Furthermore, the results presented in this study were encouraging since

editing rates were above the therapeutic threshold for both mice and non-

human primates, thus providing the basis for dose extrapolation to first-in-

human trial for a CRISPR/Cas9 gene therapy. These clinical trials would

pave the way for a new era for treating the inherited ocular diseases for

which current disease prognosis is poor.

CONCLUSION

Gene editing holds enormous potential for the clinical treatment of

inherited eye diseases. The eye, being an ideal target for gene editing puts

ophthalmology at the forefront of the emerging CRISPR/Cas technology.

Improvements in the development of more efficient delivery methods and

more precise CRISPR/Cas components will allow these therapies to become

a truly personalized option for medical treatment. Although a barrier to the

widespread clinical application of these new therapies is their financial cost,

CRISPR technology has the potential to disrupt pathology and cure genetic

disease.

In terms of cost, projections range from 500,000 US dollars to $1.5

million. The cost could limit the availability of the technology to lower

socioeconomic groups, but with future advances, the cost is likely to fall. To

prepare the insurance industry, many leaders in the field are seeking to


address the challenges, working with payers, policy makers, and other key

experts in regulatory affairs and law to recommend payment programs

(Salzman et al. 2018). There have been various models proposed to handle

the high cost of gene therapy such as a value-based payment model which

would link payments to actual outcomes observed over an extended time

period. One possibility would be for the pharmaceutical company to

reimburse the insurance provider the cost of the treatment if, after three years

there is no clinical improvement. Visual science researchers and clinicians

continue to conduct ground breaking studies that will soon translate into new

therapeutic options.

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