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Journal of Controlled Release 124 (2007) 107 – 133
www.elsevier.com/locate/jconrel
Review
Corneal gene therapy ☆
Eytan A. Klausner a,⁎, Dan Peer b , Robert L. Chapman a , Richard F. Multack c , Shridhar V. Andurkar a
a
b
Midwestern University Chicago College of Pharmacy, 555 31st Street, Downers Grove, IL 60515, United States
CBR Institute for Biomedical Research, and Department of Anesthesia, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, United States
c
Midwestern University Chicago College of Osteopathic Medicine, 555 31st Street, Downers Grove, IL 60515, United States
Received 15 January 2007; accepted 15 May 2007
Available online 4 July 2007
Abstract
Gene therapy to the cornea can potentially correct inherited and acquired diseases of the cornea. Factors that facilitate corneal gene delivery are
the accessibility and transparency of the cornea, its stability ex vivo and the immune privilege of the eye. Initial corneal gene delivery studies
characterized the relationship between intraocular modes of administration and location of reporter gene expression. The challenge of achieving
effective topical gene transfer, presumably due to tear flow, blinking and low penetration of the vector through epithlelial tight junctions left no
alternative but invasive administration to the anterior chamber and corneal stroma. DNA vaccination, RNA interference and gene transfer of
cytokines, growth factors and enzymes modulated the corneal microenvironment. Positive results were obtained in preclinical studies for
prevention and treatment of corneal graft rejection, neovascularization, haze and herpetic stromal keratitis. These studies, corneal gene delivery
systems and modes of administration, and considerations regarding the choice of animal species used are the focus of this review. Opportunities in
the field of corneal gene therapy lie in expanding the array of corneal diseases investigated and in the implementation of recent designs of safer
vectors with reduced immunogenicity and longer duration of gene expression.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Cornea; Eye; Gene therapy; Corneal graft rejection; Herpetic stromal keratitis; Corneal neovascularization
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Anatomy, physiology and immunology relevant for corneal gene therapy . . . . . .
2.1.1. Anatomy and physiology . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2. Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Vectors and methods of corneal gene therapy . . . . . . . . . . . . . . . . . . . . .
2.2.1. Viral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2. Non-viral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.3. Physical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.4. Knock-down oligonucleotides and expression cassettes, and RNA aptamers
Previous studies and relevant diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Corneal diseases and manifestations of metabolic diseases treatable by gene therapy
3.1.1. Corneal transplantation and graft rejection . . . . . . . . . . . . . . . . .
3.1.2. Herpetic stromal keratitis . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3. Corneal neovascularization . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.4. Corneal haze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
☆
This article is dedicated to the memory of Milo Gibaldi, Ph.D.
⁎ Corresponding author. Tel.: +1 630 515 7104; fax: +1 630 515 6958.
E-mail address: [email protected] (E.A. Klausner).
0168-3659/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2007.05.041
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E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
3.1.5. Mucopolysaccharidosis VII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.6. Additional proposed corneal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. In vivo corneal gene delivery studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1. Transfer of reporter genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2. Corneal transplantation and prevention of graft rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3. Prevention and treatment of herpetic stromal keratitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.4. Control of corneal neovascularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.5. Correction of corneal haze, opacity and manifestations of mucopolysaccharidosis VII, and facilitation of wound healing
3.3. Ex vivo corneal gene delivery studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Pharmaceutical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Corneal gene delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Modes of administration of corneal gene delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Animals used in corneal gene delivery studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1. Inter-species variability in the anatomy, physiology and immunology of the eye . . . . . . . . . . . . . . . .
4.3.2. The existence of an animal strain that mimics human pathology . . . . . . . . . . . . . . . . . . . . . . . .
4.3.3. The disease pattern and symptoms in the animal model in comparison to humans . . . . . . . . . . . . . . .
4.3.4. Past experience in corneal gene delivery studies of a connection between managing a specific disorder
and the use of a particular animal species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.5. The availability of analytical methods, including investigational reagents, to conduct the study in a particular animal
species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.6. Past experience in husbandry of the animals under study in research facilities . . . . . . . . . . . . . . . . .
4.3.7. Modifications and improvements of existing animal models that are suggested in contemporary publications .
5. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Corneal gene therapy initially emerged in 1994 when its
potential in correcting acquired corneal inflammatory diseases
was noted following successful transduction of corneal tissues
using replication-deficient adenovirus [1]. Inherited corneal
diseases such as corneal endothelial dystrophies [2] are natural
candidates for corneal gene therapy [3–5]. However, most of the
research has focused on modulation, including immunomodulation, of acquired medical conditions. This is feasible
since control of the corneal microenvironment can be attained by
induction or knock-down of proteins using corneal gene therapy.
Local corneal gene delivery has the potential to achieve low
and continuous concentrations of biologically active proteins
thereby improving the efficacy and safety of the treatment [6].
This methodology can be applied to the delivery of various
cytokines and growth factors to the cornea thus affecting immune
response, inflammation, angiogenesis and proliferation and cell
differentiation. Corneal gene therapy also holds promise in
obtaining local protein expression at concentrations unattainable
by systemic administration of the recombinant protein [7].
While corneal gene therapy has developed as a part of the
natural growth in the field of gene therapy, several advantages
are apparent in making the cornea particularly attractive for
gene transfer. The cornea has a well-defined anatomy [8] and is
easily accessible [8–11] during ambulatory visits, as well as
surgical procedures [12,13]. The cornea can be treated noninvasively [13] as the anatomic location of corneal epithelium
permits direct topical instillation of the gene delivery system
[14]. The perfect transparency of the cornea allows rapid and
non-invasive visual observation [8,9,15] using standard oph-
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thalmologic methods e.g., slit-lamp biomicroscopy, with high
magnification thus estimating the effectiveness and safety of the
treatment [16].
Additional factors facilitate gene therapy to both anterior and
posterior parts of the eye and are therefore relevant to corneal
gene transfer. For instance, the eye is a partially immuneprivileged site [17,18] thereby enabling the use of otherwise
immunogenic or proinflammatory vectors [18]. In terms of
experimental design, the fact that the eye is a paired organ
enables the contralateral eye to serve as an internal control
[19,20]. Also, the ocular tissues are easily anesthetized [16], and
are treatable during trabeculectomy, cataract surgery and
corneal transplantation [21]. In the same spirit, ex vivo gene
transfer to a donor cornea prior to corneal grafting entails
several advantages when compared to ex vivo manipulations of
other grafts: The cornea can be maintained in culture for up to
1 month [8,9]; its small size enables modification of the
complete tissue [18]; and the major target cell (the corneal
endothelial cell) forms a monolayer on the corneal surface and
is therefore perfectly accessible to gene vectors [7].
Due to the importance of corneal gene therapy and its
inherent advantages, studies have been conducted throughout
recent years on various types of animals to investigate new
treatments for major corneal disorders such as allograft rejection
following corneal transplantation, herpetic stromal keratitis
(HSK), corneal neovascularization and corneal haze. The potential of corneal gene therapy in decreasing the incidence of
blindness is vast, as it can enhance survival of corneal grafts as
well as ameliorate corneal disorders that currently lead to the
need for corneal transplantation thus obviating the need for an
allograft [22]. A particular challenge in corneal gene delivery
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
stems from the sensitivity of the organ in that even minimal
inflammation might impair vision [21]. This calls for extra
caution in designing the corneal gene therapy in terms of the
delivery system, mode of administration and dosing regimens.
2. Background
2.1. Anatomy, physiology and immunology relevant for corneal
gene therapy
2.1.1. Anatomy and physiology
The cornea is the avascular tissue on the surface of the eye
that is directly exposed to the external milieu, see Fig. 1A. The
cornea refracts light (with the lens), provides protection from
microscopic pathogens (with the conjunctiva and tear film) and
confers mechanical strength (with the sclera) thereby shielding
the eye from physical injuries. Regulated hydration and the
precise architecture of the cornea contribute to its unique
transparency [23] that is essential for transmittance of incident
visible light through the lens to the retina thereby enabling
vision [24].
The adult cornea is approximately 11–12 mm wide and has a
height of about 9–11 mm. Its thickness varies from 0.5 mm at
Fig. 1. A schematic of a cross section of (A) the eye, modified from [225]; and
(B) the cellular organization of the cornea, modified from [226].
109
the center to 0.7 mm at the periphery. The central 4 mm of the
corneal diameter is considered the optical area [25].
From anterior to posterior the cornea is composed of 5 layers
(see Fig. 1B): epithelium, Bowman's membrane, stroma, Descemet's membrane and endothelium. The epithelium, which is
considered “tight” [24], has a thickness of about 50 μm and is self
regenerated with a complete turnover time of 5–7 days [26]. It
protects the eye from microbes and toxins [23], prevents fluid
loss [26] and with the tear film, assists in maintaining the optical
smoothness of the corneal surface [23]. The tear film, which has a
thickness of 4–9 μm [27], lubricates the cornea and provides
nutrients and regulatory factors necessary for epithelial maintenance. Bowman's membrane is an acellular layer, 12 μm thick,
having a random arrangement of collagen fibers and proteoglycans. Its physiological role is still unclear [23].
The stroma makes up more than 90% of the corneal thickness
and is primarily composed of an extracellular matrix that is
composed of collagen and proteoglycans. The predominant
stromal cells are the keratocytes, which are corneal fibroblasts
[23] that synthesize, maintain and repair the extracellular matrix
[24] and occupy 3%–5% of the stromal volume [28]. The
unique regular arrangement of collagen fibers is a key factor in
corneal transparency [25].
Descemet's membrane, the basement membrane of the
corneal endothelium, has a thickness of 8–10 μm in adults
and is comprised of collagen and laminin, a glycoprotein. The
endothelium is a monolayer, 5 μm thick, whose major role
is the regulation of stromal hydration. On one hand the
endothelium is a “leaky” barrier to the aqueous humor due to
gap junctions between cells that allow transfer of nutrients and
electrolytes [23]. On the other hand it pumps water from the
stroma [25]. Thus, increased corneal thickness usually indicates a pathologic condition of the endothelium [28]. The
endothelium does not replicate in humans after birth, therefore
cell loss is compensated by enlargement of the remaining cells
[25].
Other relevant ocular compartments are the anterior chamber,
the vitreous body and the conjunctiva. The anterior chamber is a
compartment between the cornea and the iris that is filled with
approximately 0.2 ml of aqueous humor, which satisfies the
metabolic needs of the lens and the cornea [29]. The vitreous
body accommodates 80% of the eye's volume. It supports retinal
metabolism and serves as a diffusion barrier between the anterior
and posterior part of the eye [30]. The conjunctiva is a thin
mucous membrane that lines the inner surface of the lids [26] and
along with the cornea, forms the ocular surface [31]. In addition
to its roles in facilitating the movement of the eye and as a
reservoir for tears [32], the conjunctiva fulfils an important role in
immune responses of the ocular surface, playing host to T and B
lymphocytes, mast cells, neutrophils and dendritic cells [33].
2.1.2. Immunology
The immune response of the eye entails interactions between
local and systemic immune cells, which include macrophages,
lymphocytes, eosinophils and antigen-presenting cells; and
soluble mediators of the immune system, which encompass
complement, cytokines and immunoglobulins [34].
110
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
Despite the availability of various components for an
immune response, the ability of the eye to tolerate inflammation
is very limited, since a conventional immune response, as
observed for instance after the penetration of an antigen or a
pathogen through the skin, might destroy the microanatomy of
the visual axis needed for accurate vision. This explains the
need for a special type of immunity in the eye, termed immune
privilege [33].
Ocular immune privilege is a result of various elements.
Among these are passive elements such as the blood-ocular
barrier that physically prevents cellular infiltration [34], the
low expression of major histocompatibility complex class I
and II molecules and the lack of lymphatic drainage. Active
aspects of ocular immune privilege include the constitutive
expression of inhibitors of complement activation by corneal
endothelial cells and of Fas ligand (which induces apoptosis of
activated T cells) by corneal cells. The aqueous humor also
contributes to immune privilege as it contains immunosuppressive factors.
Another aspect of immune privilege is the eye's ability to
regulate the systemic immune response to eye-derived antigens
through a mechanism termed anterior chamber-associated immune deviation. In this case the immune response induced
by penetrating antigens is deficient in B cells that secrete
complement-fixing antibodies and in T cells that mediate delayed hypersensitivity.
The result of ocular immune privilege is that immune
defense takes place with no inflammation. To emphasize this
concept, when privilege is successful corneal allografts are
accepted and ocular infections do not inflame the eye. Failure of
immune privilege leads to the opposite and blindness is likely to
occur [33].
The cornea itself takes an active role in immune protection of
the structure and function of the eye surface in that corneal
epithelial cells and keratocytes secrete cytokines. Together with
the lacrimal gland, tear film and the conjunctiva, the cornea
constitutes the ocular mucosal-associated lymphoid tissue [34].
2.2. Vectors and methods of corneal gene therapy
Many of the prominent gene vectors, knock-down methods
and physical techniques for gene therapy have been used in
corneal gene delivery. We briefly describe these vectors and
methods.
2.2.1. Viral vectors
2.2.1.1. Adenovirus. Adenoviral vectors, commonly used in
gene therapy, have a broad range of host cells [35,36] and can
infect both dividing and non-dividing cells. Since they do not
integrate into the genome, their expression is transient [36,37].
The major hurdle in using adenoviruses for gene therapy is the
immunogenicity of their capsid and the proteins they express.
Most adenoviral vectors are derived from adenovirus serotype
5 [38], having a genome of 35 kb. The first generation
adenoviral vectors are E1-gene deleted, with partial or
complete deletion of the E3 gene [39], allowing a transgene
capacity of 8.5 kb [36]. Removal of the E1 and E3 genes
renders the virus mostly replication-deficient and increases the
vector capacity, respectively [39]. Second generation adenoviral vectors have additional deletions of part of the E2 and/or
most of the E4 genes, thereby decreasing the immune response
to viral proteins [40]. Third generation adenoviral vectors, also
termed gutless adenoviral vectors, contain only the sequences
needed for the initiation of viral DNA packaging and
replication. Therefore they are less immunogenic [39,41] and
have substantial capacity of transgene containment, N30 kb
[40].
2.2.1.2. Adeno-associated virus. Adeno-associated viruses
have a small (4.7 kb) genome, are non-pathogenic [39] and
can infect both dividing and non-dividing cells [40]. Despite the
fact that adeno-associated viral vectors do not encode for viral
proteins, a humoral immune response is elicited following their
administration due to the viral capsid [38]. In contrast to wildtype adeno-associated viruses that demonstrate site specific
genome integration, adeno-associated viral vectors integrate at
sites of double-stranded DNA breaks [39].
2.2.1.3. Retroviruses. Retroviral vectors, commonly used in
gene therapy [40], integrate into the host genome and therefore
offer long term transgene expression [37]. Due to their random
integration they might cause insertional mutagenesis i.e., insert
the transgene into a coding region [40]. For instance, as a part of
a clinical trial for the treatment of X-linked severe combined
immunodeficiency, vector integration up-regulated a protooncogene, LMO-2, leading to T-cell leukemia in 3 patients [42],
one of whom has died. Retroviral vectors can infect dividing
cells but not non-dividing cells [39].
2.2.1.4. Lentiviruses. Lentiviruses are a subtype of retroviruses that have the ability to infect both dividing and nondividing cells [37], which was one advantage of constructing
gene vectors from them [36]. The most extensively used
lentiviral vector is based on human immunodeficiency virus
type 1 [38]. A recent design of lentiviral vectors circumvented
the risk of insertional mutagenesis by integration deficiency
[43].
2.2.1.5. Herpes simplex virus-1 (HSV-1). HSV-1 is strongly
neuro-tropic [40] and can replicate in epithelial cells [36]. The
genome size of HSV-1 is 152 kb [37] and this enables HSV-1
vectors to accommodate relatively large and/or multiple
transgenes. Several types of vectors were developed based on
HSV-1: amplicon, replication defective and replication competent attenuated vectors [44]. The latter group was studied for
gene therapy for the peripheral nervous system, against cancer
and as live attenuated HSV-1 vaccines [45].
2.2.2. Non-viral vectors
2.2.2.1. Naked DNA. Naked plasmid DNA is a negativelycharged, large macromolecule with a molecular weight of
≥ 2000 kDa [46]. Naked DNA loses its stability once it enters
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
the body due to susceptibility to extracellular and intracellular
nucleases [46,47]. The characteristics of naked DNA facilitate
usage of physical techniques to improve cell entry [36], and
compounding with cationic lipids or cationic polymers to
enhance in vivo stability and cell penetration [36,46]. It is
important to note that intramuscular injection of naked DNA
used for vaccination in preclinical studies, elicited an immune
response that protected animals against infections [36]. In corneal gene therapy, naked DNA was mostly studied for treatment
of HSK, see Table 3.
2.2.2.2. Minimalistic immunologically defined gene expression (MIDGE) vectors. MIDGE vectors are double-stranded
linear, covalently closed expression cassettes that contain the
cytomegalovirus (CMV) promoter, the gene of interest and a
polyadenylation site [48]. They are less immunogenic than
standard bacterial plasmids. MIDGE vectors are not commonly used in gene therapy, although they were studied for their
utility in corneal gene therapy [48–50].
2.2.2.3. Cationic liposomes. Cationic lipids spontaneously
create complexes with negatively-charged DNA [36]. To improve the stability of the complex, which is termed lipoplex, the
cationic lipid is often combined with a neutral lipid and/or a
surfactant [47]. Creation of lipoplex protects the DNA from
degradation [51] and cationic lipoplexes facilitate penetration
via the negatively-charged cell membrane [36,51]. Cationic
lipoplexes do not elicit cellular immune response, are less
efficient than viral vectors, and have limited use in vivo due to
their inherent toxicity [47].
2.2.2.4. Polyethylenimines. Polyethylenimines, which appear
as linear or branched isomers, are the most extensively used
cationic polymers for gene delivery [52]. Polyethylenimines
spontaneously form nanoparticles with DNA [53] and have exhibited relatively high transfection efficiency in various organs
[54] although they are still less efficient than viral vectors [36].
Since polyethylenimines are considered toxic in vivo [54],
studies are ongoing to expand their in vivo potential by chemical modifications [55].
2.2.2.5. Polyamidoamine dendrimers. Dendrimers are
branched polymers with a well-defined spherical shape [56]
and very low polydispersity [57]. When using polyamidoamine dendrimers as gene carriers a higher number of layers
(‘generations’) around the core is associated with both
increased transfection efficiency and toxicity [56]; and the
in vivo transfection efficiency is comparable to polyethylenimines [58].
2.2.3. Physical methods
2.2.3.1. Electroporation. Electroporation is the enhancement of DNA penetration through the cell membrane by
creating transient and localized membrane pores using electric
fields. In vivo electroporation occurs by injecting plasmid
DNA with subsequent induction of short electrical pulses from
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electrodes located in the target tissues [36]. As an advantage,
electroporation usually increases gene expression by 100- to
1000-fold in comparison to naked DNA administration. The
disadvantage is that it is potentially harmful to the cell
membrane. Electroporation is currently being studied for gene
therapy in a wide variety of tissues including skin, skeletal
muscle, retina and cornea [59].
2.2.3.2. Gene gun. The term, gene gun, refers to the ballistic
delivery of DNA particles laden with heavy metal, usually gold,
to cells using pressure and speed [36,60]. While this approach
was most commonly used for genetic vaccination and immunomodulation of the skin, it was also studied in other tissues e.g.,
brain, liver [60], and cornea [48,61,62]. A major benefit of the
gene gun approach is the potential to obtain locally-defined
gene delivery in particular intracorneal tissues [61].
2.2.3.3. Sonoporation. Sonoporation is the enhancement of
DNA penetration through the cell membrane by creating
transient and localized membrane pores using ultrasound [60].
Sonoporation increases gene expression by 10- to 15-fold in
comparison to naked DNA administration [59] and is safe
[13,63]. It has been studied for gene delivery to solid tumors,
muscle [59] and the cornea [13]. Ultrasound contrast agents that
enhance cavitation e.g., Albunex, promote transfection [60].
2.2.4. Knock-down oligonucleotides and expression cassettes,
and RNA aptamers
2.2.4.1. Antisense oligonucleotides. Antisense oligonucleotides are typically 15–20-nucleotide [64] single-stranded DNA
sequences that hybridize in the cell to sense mRNA sequences
by reverse complementarity. The product inhibits gene
expression via several mechanisms [65]. The first antisense
oligonucleotide drug that was available in the clinical setting,
Vitravene® [64], is an intravitreal injection against CMVinduced retinitis [65].
2.2.4.2. Small interfering RNA (siRNA). Synthetic siRNA
are short, typically b 30-nucleotide double-stranded RNA sequences [66] that act in the cytoplasm of cells where they can
knock-down the expression of subsequent genes [67] more
efficiently than antisense oligonucleotides [68]. Such siRNA
sequences may be formulated for administration by various
delivery systems, and are being tested in intravenous, intraperitoneal [67] and intravitreal administrations [69] in addition
to other routes.
2.2.4.3. Short hairpin RNA (shRNA). shRNA vectors are
DNA-directed expression cassettes encoding a single RNA
palindromic sequence of 50–70 oligonucleotides that can
hybridize with itself to form a stem-loop “hairpin” structure
[70,71]. Once in the cytoplasm the structure can be cleaved to
21-nucleotide siRNA duplexes. In comparison to the latter,
shRNA viral vectors potentially offer prolonged gene suppression [70,72] and shRNA plasmid DNA vectors are easily amplified to obtain large quantities.
112
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2.2.4.4. RNA aptamers. Following synthesis by in vitro selection and amplification from a sizeable pool of RNA sequences,
RNA aptamers (ligands) bind with high affinity to small molecules and proteins, thereby potentially inhibiting the activity of
the latter [73,74].
3. Previous studies and relevant diseases
3.1. Corneal diseases and manifestations of metabolic diseases
treatable by gene therapy
Corneal gene therapy has the potential to treat and cure
inherited and acquired corneal disorders and metabolic diseases
affecting the cornea. Preclinical work in this field has concentrated on treating and preventing several acquired disorders
such as corneal graft rejection, HSK, corneal neovascularization
and corneal haze. Studies were also conducted to evaluate the
effect of systemic gene therapy on the manifestations, including
corneal, of mucopolysaccharidosis VII, a metabolic disease.
Therefore, these diseases are described below.
3.1.1. Corneal transplantation and graft rejection
Corneal transplantation, the sole treatment for many corneal
diseases that often lead to blindness [75,76], is the most commonly transplanted solid tissue [76,77] with approximately
40,000 annual transplants in the United States [75]. While corneal
transplantation success rates of 75% survival through 5 years [77]
and 60% survival through 10 years are relatively high, there is no
tendency of improvement with the passage of time [78], thereby
emphasizing the need for additional clinical solutions.
Allograft rejection occurs when a cell-mediated immune
response that involves T lymphocytes stimulates a cascade
of events that lead to graft destruction [34]. Damage to the
endothelium is crucial during this process [25]. Risk factors for
immunologic allograft rejection, which is the most common
reason for graft failure [15,76,77,79,80], include neovascularization [81,82], inflammation, glaucoma and a history of previous
graft rejection [33]. Factors that facilitate successful corneal
transplantation include the immune privilege of the tissue as well
as the site, which obviates the necessity for systemic immunosuppression or human leukocyte antigen matching, and the fact
that the donor cornea can be stored ex vivo for up to a month
before transplantation [75]. The latter is particularly appealing for
gene therapy approaches as it enables modulation of the donor
cornea prior to transplantation.
It is important to note that the majority of corneal transplantations involve full-thickness tissue replacement (penetrating keratoplasty), since this procedure produces favorable visual acuity
results [75]. Transplantation of the anterior cornea leaving the
Descemet's membrane and endothelium intact (anterior lamellar
keratoplasty), is associated with a decreased risk of graft rejection
because the endothelium is the predominant rejection site [83].
3.1.2. Herpetic stromal keratitis
There are 50,000 new cases [84] and approximately 500,000
people suffering repeated episodes of ocular HSV-1 infections
in the United States every year [85]. After the virus penetrates,
often causing a corneal epithelial infection [86], it progresses
in a retrograde manner to ganglia where it resides for the
remaining life of the host [87]. In 20% of the cases of ocular
HSV-1 infections [88] the reactivation of the virus from latency,
mostly in the trigeminal ganglion [87], leads to a viral attack on
the eye that is accompanied by an immune response
orchestrated by virus specific CD4+ T lymphocytes producing
T-helper 1 (Th1) cytokines. This leads to a variable degree of
HSK [88]. Chronic corneal inflammation associated with HSK
that involves infiltration of cells, edema, neovascularization and
scarring is potentially threatening to one's vision. Thus, HSK is
the leading infectious cause of blindness in the developed world
and is also the reason for 5–10% of penetrating keratoplasty
operations performed [89].
The current pharmacotherapeutic regimens for HSK mainly
include antiviral drugs and corticosteroids [87]. It is unequivocal that additional therapeutic approaches for treating HSK are
required [89]. To this end, efforts are underway to develop a
vaccine that will prevent a primary infection and/or recurrent
attacks of ocular HSV-1 [90]. Encephalitis as a complication of
ocular HSV-1 infection is potentially lethal in the murine model
[91,92] as well as in humans [93].
3.1.3. Corneal neovascularization
The cornea is normally avascular and corneal neovascularization, which is always pathologic [94], can occur in any layer
of the cornea [87] causing visual impairment. The sprouting of
new vessels in the cornea changes the ocular surface
microenvironment [34] and might lead to corneal opacity [94].
Corneal neovascularization plays a pivotal role in inflammation [87], which might explain the fact that neovascularization in
the host cornea is a risk factor for corneal transplant rejection
[95]. Depending on the etiology of corneal neovascularization,
traditional treatments involve the use of anti-inflammatory,
antimicrobial or immunosuppressant drugs. Clearly, there is a
need for additional therapeutic approaches [94].
3.1.4. Corneal haze
Haze is the term used to depict corneal opacity following
excimer laser procedures [96]. Following photorefractive
keratectomy most patients develop a mild haze that in the
majority of cases resolves over time [97]. Corneal haze occurs
during the wound healing process when stromal keratocytes
transform to activated fibroblasts and subsequently proliferate
and migrate to the anterior stromal compartment where they
synthesize new collagen and extracellular matrix, thus causing
opacity [98]. The current treatment for corneal haze is topical
mitomycin C, which presumably induces activated keratocyte
apoptosis. Clearly, there is a need for additional therapies [96]. It
should be noted that corneal opacity (or opacification) is the term
used to describe cloudiness due to other reasons than excimer
laser procedures, for instance corneal cauterization [99].
3.1.5. Mucopolysaccharidosis VII
The mucopolysaccharidoses are a family of inherited disorders
of a deficiency in lysosomal enzymes, involved in the degradation of glycosaminoglycans, resulting in a broad spectrum
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
of manifestations [100]. The normal corneal stroma contains
approximately 4% glycosaminoglycans and pathologic deposition
of keratan sulfate and dermatan sulfate causes corneal opacification. Mucopolysaccharidosis VII, also known as Sly disease, is a
very rare disease in which the deficiency of β-glucoronidase
causes, in addition to systemic effects, progressive corneal
clouding [101] due to the storage of glycosaminoglycans in
stromal lysosomal vacuoles [102]. This pathology might
necessitate penetrating keratoplasty [100]. No pharmacotherapy
is currently available to treat the corneal manifestations of
mucopolysaccharidosis VII [101].
3.1.6. Additional proposed corneal diseases
It was suggested in the literature that corneal gene therapy is
a promising approach for the correction of additional corneal
diseases such as corneal dystrophies [23] and in particular
corneal endothelial dystrophies, which are primary diseases
of the corneal endothelium that have an inherited component
[2]. These dystrophies encompass congenital hereditary endothelial dystrophy, Fuch's endothelial dystrophy [3–5,103] and
posterior polymorphous dystrophy [3,4]. Other diseases that
were mentioned as candidates for corneal gene therapy include
keratoconus, inherited stromal disorders [103], iridocorneal
endothelial syndrome, [2–4], recurrent anterior segment inflammation of Behcet's disease [21,104] and infectious diseases
of the cornea [5,10]. Rosenblatt and Azar further proposed
that dry eye syndrome, recurrent erosions, neurotrophic keratopathy, persistent epithelial defects and chronic epithelial
inflammatory disease can all potentially be treated by corneal
gene therapy [105].
3.2. In vivo corneal gene delivery studies
The seminal works of gene delivery to the cornea appeared in
1994–1995 and concentrated on the transfer of reporter genes
[1,3]. George et al. were the first to study the prevention of graft
rejection by ex vivo genetic manipulations of the cornea prior to
transplantation, in 1996 [8]. Additional seminal works involving corneal gene therapy were conducted by Rouse et al. to treat
HSK (1997 [88]) and Rakoczy et al. [106] to correct corneal
neovascularization.
In reviewing previous studies involving corneal gene
delivery, this article highlights the in vivo studies that use
ocular gene transfer to correct corneal diseases, as opposed to
works lacking in vivo experiments or studies using only extraocular gene transfer to manage corneal diseases. Information
regarding previous research in the area of in vivo corneal gene
delivery that includes vectors, genes, animal species, doses,
modes of administration, results of studies and references is
summarized in Tables 1–5. Each table summarizes an application of corneal gene delivery to treat a specific corneal
malady with the exception of Table 1, which describes the
transfer of reporter genes. Several corneal gene delivery studies
that involve ocular modes of administration [107–111] and
extra-ocular modes of administration [99,112–116], are not
covered in Tables 1–5. The latter studies, however, are mentioned in Section 4.
113
3.2.1. Transfer of reporter genes
The initial works that exhibited transfer of genes to the
cornea were directed at studying the ability of gene delivery
methods to transfer genes to either the eye [1] or the retina
[117]. When characterizing the ocular distribution of gene
transfer, expression was noted in the cornea, among other ocular
tissues. In 1995, Budenz et al. published the first study that
aimed mainly to examine gene delivery to the cornea and the
trabecular meshwork [3].
The use of reporter genes offers the advantages that (a) the
analytical method for the measurement of their expression is
usually sensitive and easy; (b) they lack bioactivity and are
considered generally safe, although it has been documented that
at least for several transgenes, very high expression is potentially toxic to cells; and (c) studies involving reporter genes
do not involve induction of disease that is potentially time and
effort consuming. These are some of the reasons that more
studies in corneal gene delivery were conducted by utilizing
reporter genes than using bioactive proteins for the correction of
any single corneal disease (see Tables 1–5).
Moreover, corneal gene delivery studies that use reporter
genes involve a wider array of gene vectors and methods than
corneal gene therapy studies to treat any single corneal disease.
For instance, viral vectors that were used in studies of reporter
genes that characterized gene transfer to (among other ocular
tissues) the cornea include adenovirus, adeno-associated virus,
retroviruses, lentiviruses, HSV-1 and baculovirus. The physical
methods that were used to deliver reporter genes to the cornea
are electroporation, gene gun and sonoporation.
Reporter gene delivery studies in the cornea have used many
of the most common reporter genes e.g., chloramphenicol
acetyl transferase, green fluorescent protein, enhanced green
fluorescent protein, luciferase and lacZ, which is the most
common reporter gene in corneal gene delivery (see Table 1).
The major aspects that were studied were the ability to transfer
genes to the cornea using various vectors, physical techniques
and modes of administration, ocular distribution of gene
expression, safety of gene transfer vectors and methods in the
cornea, and relationship between the dose and magnitude of
corneal gene expression. Thus, corneal gene delivery studies
that used reporter genes laid the foundation for corneal gene
therapy studies of corneal diseases.
3.2.2. Corneal transplantation and prevention of graft rejection
A summary of corneal gene therapy studies for prevention of
graft rejection appears in Table 2.
Corneal gene therapy for prevention of graft rejection is challenging since corneal transplantation is at least partially successful. In order to justify the effort, cost and risk of advancing
gene therapy research related to the prevention of graft rejection
into clinical trials, it is desirable to demonstrate prolonged life
span of the transplant and reduced treatment refractoriness leading
to rapid graft rejection in preclinical studies.
The fact that immunologic graft rejection is the major reason
for transplant failure [15,76,77,79,80] may explain the high
prevalence of corneal gene therapy studies, which aim to inhibit
the cascade of immunological events that lead to graft rejection.
114
Table 1
In vivo corneal gene delivery of reporter genes
Vector
Gene
Animal
Dosing, per eye
Mode of administration
Results a
Reference
Intracameral injection
Intravitreal injection
Intravitreal injection
Intracameral or intravitreal
injection
Topical administration
Expression in 20–30% of endothelial cells for 50 days
Expression in endothelium 1 week post-administration
Expression in endothelium 1 week post-administration
Expression in endothelium for 14 days following both
modes of administration
Naked DNA: no expression; lipoplexes: expression in
epithelium was stable for 7 days and was decreased
1 month post-administration
Topical: expression in epithelium; intracameral, intravitreal,
subretinal: expression in epithelium and endothelium;
expression was stable for 7 days and was decreased
1 month post-administration
Expression in endothelium 48 h post-administration
Without HSK: occasional expression in stroma and
epithelium; with HSK: stromal expression was
stronger than without HSK,
30 h post-administration
No expression
Expression in epithelium for 7 days
Expression in endothelium for
1 week (immunocompetent mice)
or 5 weeks (immunocompromised mice)
Expression in endothelium for
3 weeks (immunocompetent mice)
or 15 weeks (immunocodeficient mice)
No expression
[1]
[117]
[162]
[3]
Intracameral injection with
electroporation
Topical administration
Expression in endothelium for 21 days; peak on day 3
[21]
Expression in stroma 4 days post-surgery
[159]
Gene gun
Expression in epithelium 2 days post-administration
[62]
Ex vivo keratolimbal
graft transduction;
orthotopic autograft
implantation
Topical administration
with or without corneal
scarification, intracameral
or intravitreal injection
Expression in epithelium for 6 months
[166]
Mice and rats: expression in epithelium after topical
application with, but not without, corneal scarification;
rats but not mice: expression in endothelium after
intracameral and intravitreal injection; expression
declined on day 7 and ended on day 14
Expression in all layers of the cornea
Intracameral: expression in endothelium for 12 weeks;
intravitreal: no expression
[167]
lacZ
lacZ
lacZ
lacZ
Mice
Mice
Mice
Mice
3 × 10 –10 pfu, 10 μl
108 pfu/ml, 0.3–0.5 μl
5 × 106 pfu
1011 particles/ml, 2–3 μl
Naked DNA,
cationic lipoplexes
lacZ
Rats
Lipoplexes: 0.8 μg
plasmid DNA, 10 μl
Cationic lipoplexes
lacZ
Rats
0.8 μg plasmid DNA, 10 μl
Topical, intracameral,
intravitreal or subretinal
administration
Adenovirus
Naked DNA
lacZ
lacZ
Rabbits
Mice
8 × 108 pfu, 20 μl
4–100 μg, 4 μl
Intracameral injection
Topical application to
scarified cornea with or
without HSK
Naked DNA
Naked DNA
Adenovirus
lacZ
GFP
lacZ
100 μg, 4–5 μl
1 μg
4 × 106 pfu, 1 μl
Topical application
Gene gun
Intravitreal injection
Adenovirus
lacZ
2 × 106 pfu, 2 μl
Intracameral injection
Adenovirus
lacZ
Mice
Rabbits
CD-1 mice,
immunocompetent or
immunocompromised
Balb/C mice,
immunocompetent
or immunodeficient
Rats
Topical administration
Naked DNA
lacZ
Rats
1 × 103–1 × 106 pfu/ml, 5 μl,
3 times for 1 day
2.5 μg, 5 μl
MoMLV-based
retroviral vector
Plasmid DNA having
a keratin 12, corneal
epithelial specific,
promoter
MoMLV-based
retroviral vector
lacZ
Rabbits
lacZ
Rabbits
lacZ
Rabbits
HSV-1
lacZ
Mice, rats
1 × 107 pfu, 5 μl
Naked DNA
HIV-1-based
lentiviral vector
GFP
GFP
Rabbits
Mice
0.5, 5 or 50 μg/ml, 2 ml
2 × 108 transducing units/ml, 2 μl
8
100 μl, 0, 12 and 36 h
after lamellar keratectomy
2.5 μg
Iontophoresis
Intracameral or
intravitreal injection
[163]
[164]
[12]
[88]
[137]
[61]
[4]
[165]
[14]
[168]
[169]
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
Adenovirus
Adenovirus
Adenovirus
Adenovirus
7
GFP
Monkeys, n = 4
107 pfu or 109 pfu, 20 μl
Intracameral injection
Baculovirus
Adenovirus
GFP
lacZ
Mice
Rats
Intravitreal injection
Intracameral injection
Naked DNA
lacZ
Mice
106–107 infection units
1011 pfu/ml, 2 μl,
with or without cauterization
2 or 24 h post-administration
0.05–3600 ng, 2 μl
Naked DNA
EGFP
Mice
1 μg, 2 μl
Intrastromal injection
Naked DNA
GFP
Rats
0.5 μg/μl, 5 μl
Adeno-associated virus
lacZ
Rabbits
Naked DNA
GFP
Mice
107 pfu, 25 μl, followed
by inflammation induction
2 μg, 1 μl
Intrastromal injection
with electroporation
Intracameral injection
BIV-based lentiviral vector
Cationic lipoplexes,
adeno-associated virus
EGFP
CAT, lacZ
Mice
Rabbits
Naked DNA having a
CMV or keratocan
stromal specific
promoter, adenovirus
EGFP
Mice
Cationic lipoplexes
Luciferase
Rabbits
Adenovirus
5×104 or 5×105 transducing units
50 μg, 50 μl of plasmid DNA in
lipoplexes; CAT: 1 × 1011 pfu,
25 μl; lacZ: 5 × 1011 pfu, 10 μl
2 μg plasmid DNA or
1.3 × 106 pfu adenovirus, 2 μl
Intrastromal injection
Intrastromal injection
with electroporation
Intravitreal injection
Intrastromal instillation
via a lamellar flap
Intrastromal injection of
adenovirus or naked DNA
having a CMV promoter;
or subconjunctival injection
of naked DNA having
different promoters
Naked DNA: 2 μg/μl; lipoplexes: Topical instillation or
40–85 μg; 100 μl
intravitreal injection
Intracameral injection
5 × 106–6 × 107 pfu, 1–5 μl
EGFP with or
Rats
without anti-CD4
antibody fragment
Plasmid DNA with FuGENE® 6 EGFP
Rabbits
HIV-based lentiviral vector
GFP
Rats
5 μg plasmid DNA, 0.4 ml
Subconjunctival injection
103, 105, 107 and 109 pfu, 2–10 μl Intracameral injection
Naked DNA
GFP
Rabbits
10 μg, 12 μl
EIAV-based lentriviral vectors
pseudotyped with VSV-G or
rabies-G envelope proteins
EGFP
Mice
1.5 × 109–1.4 × 104 transducing
units/ml, 2 μl
Mice
5 μg, 1 μl
Intrastromal injection with
electroporation
108 pfu/ml, 10–20 μl
Intracameral or intravitreal
injection
Plasmid DNA having a CMV or Luciferase, IL-10
ubiquitin promoter
Adenovirus
Heme oxygenase-1 Rabbits
Intracorneal injection with
ultrasound and microbubbles
Intracameral or intravitreal
injection
High dose (n = 2), strong inflammation; low dose (n = 1),
no expression; low dose (n = 1), expression in endothelium
after repetitive injections
Expression in endothelium for 14 days
With or without cauterization, expression in
endothelium for 10 days
[17]
Expression in stroma and epithelium from 1 h to
5 days; peak on day 1
Expression in stroma and epithelium from 4 h to
6 days; peak on day 1
Expression in stroma during days 1–15; peak on days 2–6
[16]
Expression in endothelium was induced by inflammation
[172]
Expression in stroma and epithelium on day 3
[5]
Increased endothelial expression using a higher dose
CAT: lipoplexes, expression during 4–72 h,
adeno-associated virus, 3–7 days; lacZ: lipoplexes,
4–36 h, adeno-associated virus, 3–10 days
Expression in stroma following all
modes of administration; adenovirus: up to 21 days
[173]
[174]
Topical: no expression; intravitreal:
expression during 1–7 days; peak on day 3
Expression of EGFP in endothelium on day 3
[176]
Expression in stroma during days 3–7
Expression in endothelium 3 weeks post-administration;
higher expression was obtained for 107 and 109 pfu
Expression in stroma during days 1–8 with gradual
decrease thereafter
Intracameral: VSV-G pseudotype, expression in
endothelium for 10 months post-administration;
rabies-G pseudotype, 38% of eyes, expression in
stroma and epithelium; intravitreal: no expression
Expression mostly in stroma;
IL-10: CMV, decline after 1 day;
ubiquitin, stable for 4 days;
luciferase: ubiquitin, lasting 28 days
Intracameral: expression in endothelium and epithelium;
intravitreal: expression in endothelium
[170]
[106]
[153]
[171]
[175]
[120]
[81]
[177]
[13]
[178]
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
Adenovirus
[10]
[179]
Abbreviations: BIV, bovine immunodeficiency virus; CAT, chloramphenicol acetyl transferase; cfu, colony-forming units; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; EIAV, equine infectious
anaemia virus; GFP, green fluorescent protein; HIV, human immunodeficiency virus; HSK, herpetic stromal keratitis; pfu, plaque-forming units; IL, interleukin; MoMLV, Moloney murine leukemia virus.
a
Among various organs and ocular tissues, only expression in the cornea is described.
115
116
Table 2
In vivo corneal gene therapy for prevention of graft rejection
Gene
Animal
Adenovirus
lacZ
Rabbits
Adenovirus
TNFR-Ig
Adenovirus
CTLA4-Ig
Adenovirus
IL-4
MIDGE
IL-4, IL-10, CTLA4,
IL-4 with CTLA4
Recipients: New Zealand
white rabbits; donors:
Dutch Belted rabbits
Recipients: Lewis rats;
donors: Brown Norway rats
Recipients: Lewis rats;
donors: Wistar–Furth rats
Recipients: Balb/C mice;
donors: C3H mice
MIDGE
IL-4 with CTLA4
Recipients: Balb/C mice;
donors: C3H mice
0.1 μg, repeated twice
within 1 min
MIDGE
IL-4 with CTLA4
Recipients: Balb/C mice;
donors, C3H mice
10 μg, repeated to a total
dose of 300 μg
Adenovirus
IL-10
Adenovirus
p40 IL-12, IL-4
Adenovirus
Monomeric anti-CD4 Recipients: Fisher 344 rats;
antibody fragment
donors: Wistar–Furth rats
Lentivirus
EIAV-based
lentiviral vector
Adenovirus
Endostatin:K5
IDO
Cationic lipoplexes
or adenovirus
Viral IL-10
Recipients: Lewis rats;
Donors: Wistar–Furth rats
Adenovirus
NGF, CTLA4-Ig
Recipients: Lewis rats;
donors: Dark Agouti rats
CTLA4-Ig with or
without IL-10
Treatment
Results
Reference
Ex vivo graft transduction;
orthotopic transplantation
Ex vivo graft transduction
Expression for 4 days with no effect on corneal function
[8]
Mock adenovirus shortened survival of grafts in comparison
to TNFR-Ig adenovirus-treated or untreated grafts
[123]
1 × 109 pfu/ml, 100 μl
1 day post-transplantation
Ex vivo graft transduction
or intravenous injection
Ex vivo graft transduction
Ex vivo and systemic administrations prolonged graft survival [119]
from 9 days to 10 and 18 days (medians), respectively
No prolongation of graft survival
[9]
2 μg
All transgenes: no prolongation of graft survival over
pharmacotherapy positive control
[49]
Prolongation of graft survival from 27 ± 19 to 64 ± 28 days
[50]
Eyelid: prolongation of graft survival from 26.6 ± 5.7 to
61.7 ± 25.6 days; leg: no effect
[48]
Sheep
Glucocorticoid
pharmacotherapy with gene
gun bombardment to the
epithelium 10 days
post-transplantation
Pharmacotherapy with gene
gun bombardment to the
epithelium 1 day
pre-transplantation
Gene gun to the lower eyelid
or the leg beginning at 1 day
pre-transplantation
Ex vivo graft transduction
[18]
Sheep
Ex vivo graft transduction
Prolongation of graft survival from 20 days to
55 days (medians)
P40 IL-12: prolongation of graft survival from 20 days
to 45 days (medians); IL-4: no effect
No prolongation of graft survival
Rabbits
Recipients: Balb/C mice;
donors: C3H/He mice
Recipients: Lewis rats;
donors: Dark Agouti rats
Dosing, per eye
Injection of 2–5 × 107 pfu
Injection of 1 × 109 or
1 × 1010 pfu 1 day
pre-transplantation
Injection of 1 × 109
or 1 × 1010 pfu 1 day
pre-transplantation
Injection of 1 × 109 pfu
1 day pre-transplantation
Ex vivo graft transduction
with or without intracameral
injection to donor corneas
3 days pre-transplantation
Ex vivo graft transduction
Ex vivo graft transduction
Prolongation of graft survival from 14 and 18 days to 39 days
Prolongation of graft survival from 11 days to 21 days
(medians)
Ex vivo graft transduction
Ex vivo: prolongation from 13.1 ± 0.3 (control) to 15.8 ± 0.6
with or without intraperitoneal (CTLA4-Ig) or 15.3 ± 0.2 (CTLA4-Ig and IL-10) days;
injection
systemic, CTLA4-Ig: low dose 22.7 ± 4.3, high dose
40.4 ± 9.2 days (medians)
Ex vivo graft transduction or Ex vivo: lipoplexes or adenovirus, no effect; systemic:
intraperitoneal injection
prolongation from 10.56 ± 0.34 (control) to 14.6 ± 1.01
(low dose) days (medians); high dose, no effect
Ex vivo graft transduction,
NGF: ex vivo, prolongation from 13.1 ± 0.3 (control) to
intraperitoneal injection,
16.8 ± 1.4 days (medians); systemic, no effect; NGF ex vivo
or both
with CTLA4-Ig: ex vivo, no effect; systemic, no rejection
(6/7 animals) for 70 days post-transplantation
[118]
[120]
[82]
[76]
[15]
[79]
[80]
Abbreviations: CTLA4-Ig, cytotoxic T lymphocyte antigen 4-immunoglobulin; EIAV, equine infectious anaemia virus; IDO, indoleamine 2,3-dioxygenase; IL, interleukin; K5, kringle 5 of plasminogen; MIDGE,
minimalistic immunologically defined gene expression; pfu, plaque-forming units; NGF, nerve growth factor; TNFR-Ig, tumor necrosis factor receptor-immunoglobulin.
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
Vector
Table 3
In vivo corneal gene therapy for prevention and treatment of herpetic stromal keratitis
Vector
Gene a
Animal
Dosing, per eye
Mode of administration
Results
Reference
Intradermal injection
Subconjunctival or
intramuscular injection
No effect on HSK during 16 days post-infection
Complete survival and reduction of lesions following
intramuscular, but not subconjunctival, administration
[126]
[180]
Intraperitoneal injection
Mice
50 μg, 100 μl, 21, 42 and 63 days pre-infection
Intramuscular injection
All transgenes: complete survival and prevention of corneal
scarring 28 days post-infection
Both transgenes: complete survival and prevention of corneal
scarring 28 days post-infection
Complete survival and prevention of corneal scarring 28 days
post-infection
[127]
Mice
10 pfu, 30 and 51 days pre-infection
Subconjunctival: 100 μg, 100 μl, 14, 35, 56 and
77 days pre-infection; intramuscular: 300 μg,
14 days pre-infection
106 pfu, repeated 1 or 3 times, last or
only immunization 21 days pre-infection
106 pfu, 21, 42 and 63 days pre-infection
8
gD
gB1s
Rabbits
Rabbits
HSV-1
Mice
Naked DNA
IL-2, IL-4,
IFN-γ
IL-12p35,
IL-12p40
A cocktail of
gB, gC, gD,
gE and gI
gD, gD-IL-2
Mice
45 μg, 21 and 28 days pre-infection
Subconjunctival injection
Naked DNA
gD-IL-2
Mice
90 μg, 10 μl, 21 and 28 days pre-infection
Subconjunctival injection
Naked DNA
Naked DNA
Naked DNA
gD-IL-2
gD-IL-2
IL-2, IL-4,
IL-10, IFN-γ
IL-2, IL-4, IL-10
Mice
Mice
Mice
90 μg, 10 μl, 21 and 28 days pre-infection
90 μg, 10 μl, 21 and 28 days pre-infection
100 μg, 4–5 μl, − 3, 2 and 5 days post-infection
Topical conjunctival application
Topical conjunctival application
Topical application to scarified cornea
Mice
Topical: 100 μg, 4 μl; nasal or intramuscular:
200 μg, 25 μl; 0, 7 and 14 days pre-infection
Topical application to scarified cornea,
nasal or intramuscular administration
Antisense
oligonucleotides
Naked DNA
TNF-α
Mice
100 nM, 2 μl, − 1, 1 and 4 days post-infection
Subepithelial injection
IFN-α1
Mice
5–100 μg; − 14, − 3, − 1 or 1 day post-infection
Topical application to scarified cornea
Naked DNA
IFN-α1
Mice
Naked DNA
Naked DNA
IFN-β
IL-12, IP-10
Topical application to scarified cornea,
nasal or vaginal administration
Topical application to scarified cornea
Topical application to scarified cornea
Naked DNA
Plasmid DNA encoding
shRNA complexed
with cationic liposomes
Naked DNA
IL-18
MMP-9
Mice
Mice
(Balb/C,
GKO)
Mice
Mice
100 μg, 3 μl; topical: −1, 1 or 2 days post-infection;
nasal or vaginal: 1 day pre-infection
100 μg, 3 μl, −3, − 1 or 1 day post-infection
100 μg, 4 μl, 3 and 6 days pre-infection
100 μg, 4 μl, 2 and 4 days pre-infection
2 plasmids, each 2 μg, 1 μg/μl, 2 days pre-infection
Topical application to scarified cornea
Intrastromal injection
HSV-1
Naked DNA
Naked DNA
IL-10, IL-2,
GM-CSF
Mice
siRNA duplexes with or
without TargeTran™
Naked DNA
VEGF
Mice
IFN-α1
Mice
Naked DNA
IFN-α1
Mice
Topical: 4–100 μg, 4 μl; intramuscular: 200 μg;
9 days post-infection to animals with mild HSK
Intraperitoneal injection
Topical application to scarified cornea
(all trangenes) or intramuscular
administration (IL-10)
Subconjunctival: 10 μg, 10 μl; systemic: 40 μg, 100 μl Subconjunctival or intravenous injection
mixed with TargeTran™; 1 and 3 days post-infection
25 μg, 3 μl, 12, 24 or 48 h post-infection; repeated
Topical application to scarified cornea
3 times at 12-hour intervals
Topical application to scarified cornea
100 μg, 3 μl, 1 day post-infection
Complete survival and prevention of stromal,
but not epithelial, keratitis
Complete prevention of stromal, but not epithelial,
keratitis 10 days post-infection
Complete prevention of stromal, but not epithelial, keratitis
Complete prevention of stromal, but not epithelial, keratitis
IL-4 or IL-10: reduced lesion severity 25 days post-infection;
IL-2 or IFN-γ: no effect
Topical and nasal, but not intramuscular, administration of IL-4
or IL-10 reduced lesion severity 21 days post-infection;
IL-2: no effect
Reduction of HSK symptoms 14 days post-infection
Increased survival only when treated 1 day pre-infection using
DNA doses of 25–100 μg
Increased survival only when treated 1 day pre-infection using
topical application
Increased survival only when treated 1 day pre-infection
Balb/C: suppression of lesions using both transgenes;
GKO: IP-10, but not IL-12, suppressed lesions
Suppression of lesions 12–21 days post-infection
Suppression of lesions during 21 days post-infection
[128]
[131]
[129]
[133]
[130]
[132]
[137]
[138]
[139]
[143]
[144]
[91]
[140]
[141]
[142]
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
Vaccinia virus
Naked DNA
Topical: IL-10, but not IL-2 or GM-CSF, reduced lesion severity [88]
32 days post-infection; intramuscular: no effect
Subconjunctival and systemic administrations equally
suppressed lesions 10 days post-infection
Increased survival only when treated 12 h post-infection
[146]
[92]
Increased survival
[147]
Abbreviations: gB1s, secreted HSV-1 glycoprotein B; gD, HSV-1 glycoprotein D; GM-CSF, granulocyte-macrophage colony-stimulating factor; HSK, herpetic stromal keratitis; IFN, interferon; IL, interleukin; IP-10,
IFN-inducible protein-10; MMP-9, matrix metalloproteinase-9; pfu, plaque-forming units; shRNA, short hairpin RNA; siRNA, small interfering RNA; TNF, tumor necrosis factor; VEGF, vascular endothelial growth
factor.
a
The gene can be encoded by the vector or knocked-down.
117
118
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Table 4
In vivo corneal gene therapy for control of corneal neovascularization
Vector
Gene a
Animal Dosing, per eye
Mode of administration
Results
Reference
Antisense
ODNs
bFGF
Rabbits
Sense or antisense phosphorothioate
ODNs, but not phosphodiester ODNs,
reduced NV on day 7
[181]
Naked DNA
VEGF, sFlt-1
Mice
Implantation of a hydrogel,
pre-incubated with bFGF
and oligonucleotides, into a
stromal pocket
Intrastromal injection
[16]
Naked DNA
IL-1 RA
Mice
Adenovirus
sFlt-1
Rats
Intracameral injection
VEGF induced NV; sFlt-1 reduced NV
7 days post-administration
No effect on NV 21 days
post-administration
Reduced NV 4 days post-administration
[106]
Adenoassociated
virus
Adenovirus
sFlt-1
Rats
Intracameral injection
Reduced NV 4 days post-injury
[149]
VEGF RNA
antisense
K5
Rats
Intracameral injection
Reduced NV 14 days post-injury
[150]
Subconjunctival injection
with electroporation
Ex vivo transduction
followed by
transplantation
Implantation of pellet
into a corneal
micropocket
Intrastromal injection
Reduced NV 5 days post-administration
[151]
Reduced NV during 36 days
post-transplantation
[82]
Reduced NV 6 days post-implantation
[155]
Both transgenes reduced NV 9 days
post-administration
Reduced NV 7 and 14 days after the
last administration
[152]
Naked DNA
Lentivirus
RNA aptamer
Naked DNA
Plasmid DNA
with
FuGENE® 6
Naked DNA
Naked DNA
Rats
2 × 108 pfu/μl, 2 μl,
1 day pre-injury
50 μg, 25 μl, with
concomitant injury
Endostatin:
Rabbits
K5 fusion
gene
Angiopoietin- Rats
120 pmol, in a pellet
2
containing bFGF
Flt23K,
Flt24K
BAI1-ECR
IL-12, IP-10
IL-18
Mice
Mice
Plasmid DNA
encoding
shRNA
complexed
with cationic
liposomes
Naked DNA,
albumin
polyplexes
MMP-9
Mice
Flt23K
Mice
Cytochrome
P4504B1
PEI polyplexes, bFGF
dehydrated
1 μg, 2 μl, 2 days pre-injury
Rabbits 5 μg, 0.4 ml, 2 or 3 doses
with 1-week interval,
1 week post-injury
Mice
100 μg, 4 μl, 3 and 6 days
before HSV-1 infection
Mice
100 μg, 4 μl, 2 and 4 days
before HSV-1 infection;
1 μg, 2 μl, 1 day before
CpG ODN pellet implantation
siRNA duplexes VEGF
with or
without
TargeTran™
Naked DNA
1.2 μg, 2 μl, sFlt-1:
1 day before NV challenge
1 μg, 2 μl, with
concomitant injury
1011 pfu/ml, 2 μl, 2 or
24 h pre-injury
4 × 1011 pfu/ml, 2 μl,
3 weeks pre-injury
Subconjunctival injection
Topical application
to scarified cornea
HSV-1 challenge: topical
DNA application to
scarified cornea; CpG
challenge: intrastromal
DNA injection
Subconjunctival or
intravenous injection
Subconjunctival: 10 μg, 10 μl;
systemic: 40 μg, 100 μl mixed
with TargeTran™; 1 and 3 days
after HSV-1 infection;
subconjunctival 1 day or
systemic 6 and 24 h after
CpG ODN pellet implantation
2 plasmids, each 2 μg, 1 μl,
Intrastromal injection
2 days before HSV-1 infection
Polyplexes: 2 μg, 2 μl,
3 weeks pre-injury
Rabbits 2 μg, 4 μl
Rats
Intrastromal injection
0.1, 1, 10 μg
[153]
[81]
Both transgenes reduced NV
[140]
during 20 days post-infection
HSV-1 challenge: reduced NV 9–21 days [141]
post-infection; CpG challenge: reduced NV
4 and 7 days post-implantation
HSV-1 challenge: both administrations
equally reduced NV 10 days
post-infection; CpG challenge:
both administrations reduced NV
4 and 7 days post-implantation
[146]
Reduced NV during 21 days
post-infection
[142]
[154]
Intralimbal injection
Polyplexes, but not naked DNA,
reduced NV 14 days
post-administration; expression
of Flt23K for 8 days (naked DNA)
or 5 weeks (polyplexes)
Induced NV 3 days post-administration
Corneal pocket
implantation
Induced NV; peak on days 15–21 (0.1 μg), [156]
18–24 (1 μg) and 24–30 (10 μg)
Intrastromal injection
[157]
Abbreviations: BAI1-ECR, brain-specific angiogenesis inhibitor 1 – extracellular region; bFGF, basic fibroblast growth factor; HSK, herpetic stromal keratitis; HSV,
herpes simplex virus; IL, interleukin; IL-1 RA, interleukin-1 receptor antagonist; IP-10, IFN-inducible protein-10; K5, kringle 5 of plasminogen; MMP-9, matrix
metalloproteinase-9; NV, neovascularization; ODN, oligodeoxynucleotide; PEI, polyethylenimine; pfu, plaque-forming units; sFlt-1, soluble Flt-1; shRNA, short
hairpin RNA; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.
a
The gene can be encoded by the vector or knocked-down.
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119
Table 5
In vivo corneal gene therapy for correction of corneal haze, opacity and manifestations of mucopolysaccharidosis VII, and facilitation of wound healing
Vector
Mode of administration
Results
MoMLV-based HSVtk
retroviral
vector
Gene
Animal Dosing, per eye
Topical application
Retroviral
vector
Topical application
Combined administration with
[159]
ganciclovir, but not ganciclovir alone,
reduced corneal haze 10 and 21 days
post-administration
Reduced corneal haze for 2–4 weeks post- [98]
administration
Adenovirus
Rabbits 100 μl, applied every 4 h for
2 days following lamellar
keratectomy; combined
with ganciclovir treatment
dnG1
Rabbits 5 × 107 cfu/ml, first day
following phototherapeutic
keratectomy: 40 μl every
10 min for 2 h; second day:
similar treatment for 1.5 h
tPA
Rats
5 μl, concomitant with
induction of fibrin formation
β-glucoronidase Mice
1010 pfu/ml, 0.3–1 μl
Adenovirus
β-glucoronidase Mice
5 × 107 pfu; intracameral:
20 μl, intrastromal: 50 μl
Plasmid DNA
OGFr
Rats
2 μg
Plasmid DNA OGFr
encoding for
OGFr sense
or antisense
Rats
2 μg, 1 day pre-injury
Naked DNA
Intracameral injection
with electroporation
Intravitreal injection
Reduced corneal opacity 3 days
post-treatment
Incomplete phenotypic correction of
MPS VII-related storage vacuoles in
endothelium, but not stroma, 3 weeks
post-administration
Intracameral injection;
Intracameral and intrastromal: reduction
or intrastromal sponge
and almost complete abolition of MPS
soaked with viral solution VII-related storage vacuoles, respectively,
following lamellar
5 days post-treatment
keratotomy
Gene gun
Reduced DNA synthesis in peripheral
epithelium 24 h post-administration
Gene gun
OGFr sense delayed wound healing 16,
24 and 28 h post-injury; antisense
accelerated wound healing 16 and 24 h
post-injury
Reference
[6]
[162]
[102]
[160]
[161]
Abbreviations: cfu, colony-forming units; dnG1, dominant negative mutant cyclin G1; HSVtk, herpes simplex virus thymidine kinase; MoMLV, Moloney murine
leukemia virus; MPS, mucopolysaccharidosis; OGFr, opioid growth factor receptor; pfu, plaque-forming units; tPA, tissue plasminogen activator.
Since elevated levels of Th1 cytokines were found in rejected
corneal grafts and Th1 and T-helper 2 (Th2) cells are mutually
inhibitory [9], various corneal gene therapy studies were conducted to support the Th2 and/or inhibit the Th1 pathway. For
example, studies investigated the corneal gene transfer of IL-4
[9,48–50,118] and IL-10 [15,18,49], which are Th2 cytokines,
and of cytotoxic T lymphocyte antigen 4-immunoglobulin
(CTLA4-Ig) [15,49,119] and p40 IL-12 [118], which are
antagonists of Th1 response.
Additional immunomodulatory molecules transferred to corneal grafts as genes include a monomeric anti-CD4 antibody
fragment [120]; CTLA4 [48,50], a glycoprotein on the surface of
T cells that down-regulates their activation [121]; viral IL-10
[79], a homolog to the human IL-10 that exhibits the immunosuppressive properties but lacks the immunostimulatory
effects of human IL-10 [79,122]; indoleamine 2,3-dioxygenase,
an intracellular enzyme that arrests activated T cells in the G1
phase [76]; and tumor necrosis factor receptor-Ig, a proinflammatory cytokine and inhibitor of tumor necrosis factor [123].
A different approach to prolonging corneal graft survival is to
directly inhibit the activation of endothelial cells and neovascularization processes that are related to graft rejection. Stout et al.
studied the corneal gene delivery of endostatin:kringle 5 of
plasminogen, a fusion protein that prevents endothelial cell proliferation and migration and also has anti-angiogenic properties.
Lentiviral-vector transduction of endostatin:kringle 5 in rabbits
prolonged graft survival from 14 and 18 days in the negative
control group to at least 39 days [82]. A comprehensive review of
graft failure processes that are potentially susceptible to gene
therapy can be found elsewhere [124].
The transgenes used to prevent corneal graft rejection often
encode for proteins previously shown to prolong corneal graft
survival upon administration. Using such transgenes improves
the chances of favorable outcomes in the extension of graft
survival. For example, tumor necrosis factor receptor-Ig [123],
CTLA4-Ig [119] and anti-CD4 antibodies [120] prolonged graft
survival when administered as proteins in preclinical studies. The
rationale for conducting subsequent gene delivery studies was the
anticipation that gene therapy would extend graft survival for
longer time spans than protein therapy. The advantages of gene
therapy over protein therapy are well known and include benefits,
such as continuous and stable protein levels, that should improve
the efficacy of the treatment [10,125] and result in fewer side
effects [6]. However, unless the needs for optimization of the
delivery system, mode of administration and dose are met, it is
possible that protein delivery will yield prolonged corneal graft
survival in comparison to gene transfer. Such a result was
obtained when Larkin et al. incubated de-epithelized donor
rat corneas ex vivo with CTLA4-Ig or adenovirus encoding
CTLA4-Ig prior to transplantation [119].
The outcomes of corneal gene therapy studies for prevention
of graft rejection emphasize the challenges in achieving this
goal. Several studies were unable to prolong graft survival
[9,120]. Williams et al. were able to extend median graft survival
time in sheep from 20 days to 45 [118] or 55 [18] days. However,
in both studies the authors expressed their concerns that several
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animals were refractory to the treatment and rejected the graft
in similar time periods as untreated control group [18,118].
Hoffmann et al. demonstrated extension of graft survival using
gene gun from 22.5 ± 1.7 (mean ± standard deviation) days in
the negative control group to 42 ± 21.5 days in the combined
treatment of glucocorticoid with IL-4 and CTLA4 gene transfer.
However, the extension was not significantly longer than the
positive control group, which received only glucocorticoid
treatment that resulted in graft rejection after 34.6 ± 7.8 days
[49]. This result highlights the importance of using a positive
control in such studies.
A subsequent study by Hoffmann et al. combined pharmacotherapy of dexamethasone eye-drops with gene gun IL-4 and
CTLA4 transfer, using a different DNA dose and administration
time compared with the previous study, thereby extending graft
survival from 27 ± 19 days in dexamethasone positive control
group to 64 ± 28 days in the treated group. An additional
outcome of the study was that more aggressive treatments do not
necessarily improve, and might be detrimental to graft survival.
For example, gene gun treatment of recipient mice on day (− 1)
prolonged graft survival, while a double treatment of recipient
mice on days (− 1) and 2 post-transplantation, or treatment of
both recipient and donor mice on day (− 1), did not [50].
When conducting corneal gene therapy studies for prevention
of graft rejection, one should be aware that the use of vectors and
physical methods of corneal gene therapy might have a detrimental effect on corneal graft survival. This was demonstrated
when mock adenovirus, which did not encode for therapeutic
transgenes, significantly shortened graft survival in comparison
to the negative control group or the group treated with adenovirus encoding for tumor necrosis factor receptor-Ig [123].
3.2.3. Prevention and treatment of herpetic stromal keratitis
A summary of corneal gene therapy studies for prevention
and treatment of HSK appears in Table 3.
In this section, in addition to the description of gene therapy
studies for the control of stromal disease resulting from HSV-1, we
also briefly review works describing corneal gene transfer, where
the major therapeutic index for treatment success was the ability to
protect mice from ocular HSV-1-induced encephalitis. These studies, by Carr et al., are relevant for HSK and corneal gene therapy.
There are several potential strategies to control HSK that
corneal gene therapy can address: (a) vaccination against a
primary ocular HSV-1 infection; (b) prevention of recurrent
episodes of viral attacks on the cornea and/or the accompanying
immune response due to an existing HSV-1 infection; and
(c) treatment of existing HSK symptoms.
Corneal gene therapy studies were conducted for prevention
of a primary ocular HSV-1 infection and HSK using gene-based
vaccination against viral envelope glycoproteins. Recombinant
vaccinia virus was studied by Nesburn et al. [126]. Recombinant
HSV-1 type viruses that encode for cytokine adjuvants were
studied by Ghiasi et al. [127,128]. Naked DNA vaccination and
the effect of DNA-encoded cytokine vaccine adjuvants on the
prevention of HSK were studied by Inoue et al. [129,130].
Ghiasi et al. also studied multigenic naked DNA vaccination
[131]. In all of these studies the experimental animals were
challenged with ocular HSV-1 at least 21 days after the last
treatment of an immunological boost.
HSV-1 glycoprotein D (gD) is a viral glycoprotein that was
used either as a single glycoprotein or as a part of a cocktail of
glycoproteins, in most of the gene-based vaccination studies
against infection of ocular HSV-1 and for prevention of corneal
scarring and HSK [126,129–133]. This glycoprotein facilitates
the binding and entry of the virus into the cell, is extensively
expressed by the virus [85], and was identified in previous
vaccination studies as more effective than other glycoproteins in
protecting animals from HSV [129].
The development of a multigenic vaccine encoding 5 glycoproteins by Ghiasi et al. was based on previous studies, which
showed that vaccines against only a single HSV-1 glycoprotein
were not as successful in protecting the host from eye disease as
vaccines against a cocktail of ≥5 glycoproteins [85]. A suggested
explanation was that a cocktail of glycoproteins enhances the
efficiency of the immunization against a range of variants and
strains of HSV-1. This vaccine was injected intramuscularly as
naked DNA and protected mice from corneal scarring and ocular
HSV-1-induced encephalitis [131].
The use of naked DNA vaccination in corneal gene therapy
studies protected experimental animals from HSK [129,130,
132,133], corneal scarring [131] and ocular HSV-1-induced
encephalitis [129,131]. Naked DNA vaccination is potentially
advantageous in comparison to a different contemporary biotechnological approach, of recombinant protein-based vaccination. The pharmaceutical production of naked DNA vaccines is
considered easier in comparison to recombinant proteins [125].
While recombinant protein vaccination elicits mostly humoral
immune response, naked DNA vaccination also induces cellmediated immune responses, which are often instrumental for
effective vaccination [134]. A study on the effect of administration of recombinant protein or DNA encoding gD-IL-2 (a fusion
protein of gD and IL-2) for vaccination against ocular HSV-1
using subconjunctival injection in mice, showed that both treatments significantly reduced HSK symptoms 10 days following
HSV-1 infection [133].
Live recombinant viruses [127,128] and naked DNA vaccines
[129,130,132,133] against ocular HSV-1 and HSK manifestations, used DNA cytokine adjuvants such as IL-2 [127,129,
130,132,133], IL-4, IFN-γ [127], IL-12-p35, and IL-12-p40
[128]. Cytokine adjuvants are used to improve the cytokine
environment for expansion of CD4+ and CD8+ T lymphocytes
during vaccination [135]. An advantage of utilizing cytokine
DNA over cytokine protein is that the latter has a very short halflife, a limitation that can be circumvented by gene therapy [136].
In corneal gene delivery studies, the use of cytokine DNAs
did not affect therapeutic indices such as protection from
HSK symptoms, corneal scarring and HSV-1-induced encephalitis, as the vaccinations yielded optimal results even without
the adjuvants [127–129]. However, Inoue et al. reported that
immunization with gD-IL-2 enhanced cytotoxic effector cell
activity and delayed type hypersensitivity response significantly
more than gD immunization without IL-2 [129]. These results
were the basis for the use of gD-IL-2 in their subsequent studies
[130,132,133].
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
Designing a therapeutic vaccine for the prevention of recurrent HSK episodes is desirable [85,90]. Corneal gene therapy
studies to achieve this goal have not yet been conducted.
Vaccination during the latent phase of ocular HSV-1 infection
requires modulation of the immune response and is considered
even more challenging than prophylactic vaccination against a
primary infection of ocular HSV-1 [90].
Attempts to attenuate HSK and corneal symptoms in a
relatively early stage of the disease progression, were conducted
by Rouse et al. [137,138] and Heiligenhaus et al. [139] utilizing
gene-based immunomodulation, and by Rouse et al. [140–142]
using anti-angiogenesis. In all of these studies at least part of the
treatment occurred ≤3 days prior to ocular HSV-1 infection.
This approach does not involve immunological memory and is
potentially effective only for the short term. Therefore such
treatments cannot be applied in the clinic for prevention of a
primary infection and are more relevant to the prophylactic
treatment of recurrent disease. Corneal gene transfer studies that
involve treatment shortly before HSV-1 challenge were also
conducted by Carr et al. [91,143,144].
The pathology and tissue destruction of HSK is related to the
host immune response to HSV-1 infection that is CD4+ T cell
orchestrated and typically involves cytokines of the Th1 type
[88], while later appearance of Th2 cytokines is probably
associated with lesion reduction [92,145]. This explains the
immunomodulation approach underlying gene transfer studies
to treat HSK. For example, genes for IL-10, which partially
inhibits the Th1 type pattern and was observed in corneas
during lesion resolution [88], and IL-4, which modulates the
immune response toward the Th2 pattern [137,138], were
administered as naked DNA. Tumor necrosis factor-α, a Th1
cytokine [89] that is also a key HSK mediator, was knockeddown using administration of antisense oligonucleotides [139].
Neovascularization is an essential process in HSK that
facilitates the infiltration of CD4+ T cells to the stroma. The
discovery, during a gene transfer study related to immunomodulation of HSK, that IL-12 can suppress corneal lesions via an
anti-angiogenic mechanism [140] led to the evaluation of
additional anti-angiogenic approaches for the treatment of HSK.
These include administration of naked DNA encoding IL-18, an
anti-angiogenic factor [141], and the use of RNAi to knockdown the expression of vascular endothelial growth factor
(VEGF) [146] or matrix metalloproteinase-9 (gelatinase B)
[142], which are known angiogenic factors.
The importance of the timing of corneal gene transfer for the
protection from ocular HSV-1-induced encephalitis, in relation to
HSV-1 infection was highlighted in the studies of Carr et al. that
utilized gene delivery of IFNs type 1 (e.g., IFN-α1 and IFN-β),
which are potent antiviral cytokines [91]. A treatment of the
experimental animals that used a single dose of 100 μg naked
DNA 1 day prior to HSV-1 infection increased survival, while
treatments 14 or 3 days pre-infection and 1 or 2 days post-infection
did not [91,143,144]. A different treatment of 3 doses of 25 μg
naked DNA administered in 12-hour intervals was effective when
it was administered 12 h, but not 24 or 48 h, post-infection. It is
interesting to note that while all of these studies used an HSV-1
challenge of 450 plaque-forming units, a separate study showed
121
that by using a decreased HSV-1 challenge of 150 plaque-forming
units with a similar single dose of 100 μg naked DNA, even a
treatment 1 day post-infection can increase survival of animals
[147].
A study by Rouse et al. stands out in that the treatment took
place 9 days post-infection using experimental animals, which
were pre-selected as presenting a mild [88], although not peak
[140], HSK manifestations [88]. This study, which utilized DNA
immunomodulation, better mimics a clinical situation where the
treatment aims to suppress existing HSK lesions. Additional
studies that are relevant to post-infection treatment were
conducted by Rouse et al. using an anti-angiogenic approach
[146] and by Carr et al. [92,147].
3.2.4. Control of corneal neovascularization
A summary of corneal gene therapy studies for control of
corneal neovascularization appears in Table 4.
Corneal neovascularization is usually caused by infectious,
inflammatory or traumatic conditions [148]. In corneal gene delivery studies aiming to suppress neovascularization, animal subjects were pre-treated to induce corneal neovascularization
by various methods such as chemical injury [106,149–151],
mechanical injury [81,152] or both [153,154].
Since corneal neovascularization is a central component in
the development of several corneal diseases, other corneal gene
therapy studies investigated the potential of treating the disease
by reducing neovascularization that is induced as a part of
disease progression. For example, following corneal transplantation the delivery of an anti-angiogenic fusion gene, endostatin:K5, inhibited neovascularization and graft rejection [82].
Rouse et al. studied the ability of anti-angiogenic corneal gene
delivery to inhibit HSK and the progression of neovascularization progression following HSV-1 challenge [140–142,146].
Most of these studies also included experiments evaluating
similar methods in a different corneal model where neovascularization was induced by implantation of a pellet that contains
VEGF [140] or CpG-containing oligonucleotides [141,146],
which are inducers of VEGF expression [146].
Levels of VEGF, the primary mediator of neovascularization
in the eye [149], were found to be elevated in inflamed and
vascularized corneas of rats and humans [150]. This may explain
the fact that reduction of VEGF levels is the most common
approach for inhibiting corneal neovascularization in corneal gene
therapy studies. For example, the gene delivery of a secreted form
of Flt1, a VEGF receptor [106], was studied using naked DNA
[16], adenovirus [106] and adeno-associated virus [149]. Naked
DNA encoding for Flt23K or Flt24K, VEGF-binding domains of
Flt1 fused with KDEL (an endoplasmic reticulum retention
peptide), or albumin polyplexes as vehicles for Flt23K DNA
[154], were used for intracellular inhibition of VEGF [152].
RNAi [146] or adenovirus encoding VEGF RNA antisense [150]
were used for VEGF knock-down studies. Gene transfer of IL18, a known anti-angiogenic factor, suppressed corneal neovascularization and reduced VEGF expression in vitro and in vivo
[141]. An RNA aptamer that inhibits activity of angiopoietin-2,
an enhancer of VEGF angiogenic activity, suppressed neovascularization when implanted, after compounding with a basic
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fibroblast growth factor-containing pellet, in a corneal micropocket [155]. Naked DNA administration of IL-1 receptor
antagonist that was shown in a model of penetrating keratoplasty
to reduce VEGF expression was unable to affect VEGF
expression in a model of corneal neovascularization [153].
Other corneal gene therapy studies have used transgenes
encoding for known anti-angiogenic factors from other disease
models, biological systems and target organs [81,82,140,151].
It is important to note that while the majority of corneal gene
therapy studies for control of neovascularization aim to reduce
neovascularization, a few of these studies concentrate on induction of the condition. For instance, in order to study the
potential of gene therapy by the administration of naked DNA
to improve the treatment of corneal diseases, Adamis et al.
examined the induction of neovascularization using naked DNA
encoding for VEGF [16]. Kuo et al. studied the ability of a
dehydrated form of a polyethylenimine–DNA complex to induce
corneal neovascularization using DNA encoding for basic
fibroblast growth factor. The cornea was used as a model due
to factors facilitating the use of biomicroscopy in following
neovascularization processes, such as accessibility and visibility
[156]. In order to study the importance of the cytochrome
P4504B1 mediated pathway for future anti-angiogenic treatments of corneal neovascularization, Laniado-Schwartzman et al.
investigated the ability of naked DNA encoding for cytochrome
P4504B1 to cause corneal neovascularization [157].
3.2.5. Correction of corneal haze, opacity and manifestations of
mucopolysaccharidosis VII, and facilitation of wound healing
A summary of corneal gene therapy studies to treat corneal
haze, opacity and manifestations of mucopolysaccharidosis VII
and facilitate wound healing appears in Table 5.
The corneal clouding that gene therapy studies aim to correct
may result from excimer laser procedures, fibrin formation and
mucopolysaccharidosis VII. The difference in the etiologies,
however, leads to disparity in the pharmacological target of the
treatment.
Corneal gene therapy studies were conducted to inhibit the
development of corneal haze related to excimer laser procedures
by inducing apoptosis of activated keratocytes. The transgenes
used were dominant negative mutant cyclin G1, a survival factor
that triggers apoptosis in various proliferative cells [98], or a
suicide gene/prodrug combination of a retrovirus encoding the
HSV thymidine kinase gene with ganciclovir [159]. HSV
thymidine kinase transforms ganciclovir to a toxic metabolite in
proliferating keratocytes thereby triggering cell death [158,159].
Fibrin formation might lead to corneal opacity. A previous
study was conducted to examine the potential of recombinant
tissue plasminogen activator, a fibrinolytic enzyme, to treat
fibrin clots in the anterior chamber of the eye. However, side
effects associated with the protein administration prompted the
rationale for a corneal gene therapy study that involved the
transfer of a tissue plasminogen activator gene [6].
Corneal gene therapy studies were also performed to correct
the corneal clouding manifestations of mucopolysaccharidosis
VII. Interestingly, this is the only inherited disease with corneal manifestations that was subject to corneal gene delivery,
and the transfer of the β-glucoronidase gene was hoped to
compensate for the metabolic defect. The seminal work of Li
and Davidson focused on retinal symptoms of mucopolysaccharidosis VII, recording no significant effect of the treatment on the cornea [162]. A later study concentrated on the
cornea and noted substantial reduction of storage vacuoles in
keratocytes [102].
Corneal gene therapy studies investigated the role of the
opioid growth factor receptor pathway in the regulation of cell
proliferation [160] and, in a subsequent study, wound healing
[161] in the corneal epithelium. The rationale for the studies was
based on previous works, which documented that the administration of opioid growth factor decreased DNA synthesis and
delayed wound healing [160].
3.3. Ex vivo corneal gene delivery studies
The ex vivo studies of corneal transfection were often used
by researchers prior to in vivo evaluations of corneal graft
transplantation to optimize the genetic modification of the
donor cornea. In other cases they were simply used as a
complementary to an in vitro experiment since the ex vivo
setting better reflects the in vivo setting. Table 6 lists the vectors,
genes and mammalian sources of cornea, which were used in ex
vivo studies. Reports of studies that include both ex vivo and in
vivo studies are not mentioned in Table 6.
In a particularly interesting ex vivo study, George et al. used
adenoviral vectors to deliver the lacZ or the CTLA4-Ig genes
to human corneas. CTLA4-Ig is a fusion protein that has
previously extended corneal graft survival in rats and mice. The
β-galactosidase transgene was expressed solely in the endothelium, probably since the endothelium or Descemet's membrane
acted as a barrier to adenoviral infection of the stroma.
Expression of lacZ and of CTLA4-Ig reached relatively high
levels at 7 days and 28 days, respectively. It was suggested that
the difference between the transgenes might be attributed to
different kinetics of expression of secreted versus cytoplasmic
proteins. Interestingly, mRNA of lacZ was detectable for only a
short period of 3–7 days, despite existence of its DNA in the
cell for at least 56 days. It is possible that the promoter was
inactivated during that time [182].
4. Pharmaceutical aspects
4.1. Corneal gene delivery systems
An ideal corneal gene delivery system would effectively
induce gene expression in an amplitude, time span and intracorneal tissue according to the clinical need. It would be nontoxic, non-immunogenic and would have the ability to transfer
multiple genes, regardless of their size. Also desirable is a
potential for large scale, reproducible production [38] leading to
a stable pharmaceutical product.
The most common ophthalmic drug delivery system, topical
eye-drops [203], would also be a convenient delivery system of
genes to the cornea. Advantages of eye-drops over petrolatumbased ointment, another common ophthalmic delivery system,
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
Table 6
Studies involving ex vivo gene delivery to the cornea
Vector
Gene
Adenovirus
Adenovirus with RSV or
CMV promoter
Complexes of plasmid DNA
with mollosin/polylysine peptide
Cationic lipoplexes with or
without adenovirus that does
not encode for a transgene
(lipoadenofection), with an
optional E-selectin promoter
in the plasmid DNA
Complexes of plasmid DNA,
integrin-targeting peptide and
lipofectin
Polyamidoamine dendrimers
Adenovirus
HIV-1 based lentiviral vector
Adeno-associated virus, HSV-1
lacZ
Rat, rabbit
lacZ, CTLA4-Ig Human
VP1 pseudocapsids from murine
polyoma virus
Cationic lipoplexes that include
transferrin (optional)
Polyplexes with transferrin–
polyethylenimine conjugate,
FuGENE 6 or TransIT-LT
Adenovirus
Lipoplexes optionally containing
mABs against human transferrin
receptor or E-selectin
Cationic lipoplexes, adenovirus
Cationic lipoplexes
Complexes of plasmid DNA
with mollosin/polylysine peptide
EIAV with an inducible E-selectin
promoter and HIV-1-based
lentiviral vectors
Adenovirus
Adenovirus
Adeno-associated virus
HIV-1-based lentiviral vector
lacZ
Source
of cornea
Reference
[183]
[182]
lacZ, CAT
Rabbit,
[184]
pig, human
Rabbit
[185]
lacZ
Rabbit
[186]
lacZ, TNFR-Ig
IL-4
EGFP
lacZ
Rabbit
Rat
Human
Rabbit,
human
Rabbit
[187]
[188]
[189]
[190]
lacZ, viral IL-10 Rabbit,
human
EGFP
Rabbit
[192]
Human catalase
lacZ
Rabbit
Human
[194]
[195]
Viral IL-10
EGFP, E2F2
lacZ
Human
Rabbit
Rabbit
[122]
[196]
[197]
lacZ, EGFP
Rabbit,
human
[198]
EGFP, E2F2
EGFP, scFv
GFP
EYFP
Human
Human
Human
Rat, sheep,
human
[199]
[200]
[201]
[202]
lacZ
[191]
[193]
123
Only a few studies of corneal gene transfer utilized gene
delivery systems other than a standard solution. In one such
study an intrastromal lamellar pocket was created in rats to
implant a dehydrated form of a polyethylenimine–DNA
complex. The dual advantage of the implantation is the control
over the dose as well as the anatomical site of transfection [156].
A different study extended the contact time of the viral vector
with the murine stroma to a few minutes by inserting a sponge
soaked with 50 μl of the viral solution to a wound created by
lamellar keratotomy [102].
Many of the traditional DNA vectors of gene therapy have
been tested in corneal gene transfer (see Tables 1–5). These
include viral vectors such as adenovirus, adeno-associated virus,
lentiviruses and retroviruses, and the non-viral vectors of naked
DNA, cationic lipoplexes, polyethylenimine and MIDGE. Recently the use of RNAi duplexes [146] and a plasmid DNA
encoding shRNA in the cornea were reported [142]. Generally,
viral vectors find substantially more common use in gene therapy
than non-viral vectors [36,40]; however, corneal gene delivery
studies do not follow that pattern, as viral vectors were used in
approximately 47% of the studies, see Fig. 2.
The choice of the vector is a major factor in the strategy to
rationally design a corneal gene delivery system. Various vectors
have different inherent traits and past experiences, both inside and
Abbreviations: CAT, chloramphenicol acetyl transferase; CMV, cytomegalovirus;
CTLA4-Ig, cytotoxic T lymphocyte antigen 4-immunoglobulin; EGFP, enhanced
green fluorescent protein; EIAV, equine infectious anaemia virus; EYFP, enhanced
yellow fluorescent protein; GFP, green fluorescent protein; HIV, human immunodeficiency virus; HSV, herpes simplex virus; IL, interleukin; mAB, monoclonal
antibody; RSV, rous sarcoma virus; TNFR-Ig, tumor necrosis factor receptorimmunoglobulin.
are their ease of use and the fact that they do not blur the vision
[204]. An additional lesson to learn from ocular drug delivery
systems in the development of corneal gene delivery systems
relates to the concentration of the formulation. It is preferable to
adjust the dosing to a small drop size of 5–15 μl [203] compared
to a standard drop size of approximately 50 μl [205] because
higher volumes result in spillage of the solution over the lid
margin onto the skin, due to the blinking reflex [204]. The small
drop size is related to the fact that the tear film volume is 7–
10 μl [27] and the eye can momentarily hold up to 30 μl [205].
Fig. 2. The gene vectors and methods used in corneal gene delivery studies in
vivo (1994–2006). Data were compiled from studies mentioned in Tables 1–5,
studies involving extra-ocular modes of administration [99,112–116] and
additional studies [107,109–111]. For studies that involved experiments with
two types of vectors [146,163,174,175], both vectors were taken into account.
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outside the cornea, can assist in this decision. Some components
of vectors can also confer tissue selectivity and enable activation
and deactivation of gene expression. Rational design of the
delivery system does not obviate the need to optimize the DNA
dose as was demonstrated by corneal gene delivery studies.
The importance of understanding the inherent characteristics
of the vector for the design of corneal gene delivery systems was
exemplified when the inability of retroviruses to transduce nondividing cells [39] highlighted the need to test other gene vectors
for corneal gene therapy. This was due to the hypothesis that
retroviral vectors are not expected to be useful in transducing
human corneal endothelium [3,159] and stroma [171].
Consistent with previous findings, viral vectors are generally
more efficient than the non-viral vectors in transducing their
genes [38] to the cornea in vitro and in vivo. For instance,
adenovirus was substantially more efficient than cationic lipoplexes in producing expression of the lacZ gene in ovine
corneal endothelial monolayers [206]. Following administration
to rabbit stroma via a lamellar flap, adeno-associated virus
yielded higher expression of the lacZ gene and chloramphenicol
acetyl transferase in keratocytes than cationic lipoplexes [174].
In comparison to naked DNA, administration of polyplexes
offers the potential advantages of enhancing the transgene
expression [207] and the biological effect [208]. These traits
were recently demonstrated in corneal gene therapy when
intrastromal injection of albumin-DNA nanoparticles extended
Flt23K expression and substantially reduced the development
of neovascularization [154].
Tissue selectivity can be achieved by the use of tissuespecific promoters that can be cloned into the viral vector or the
plasmid DNA. This methodology has been successful in
obtaining exclusive gene expression in corneal stroma [175]
and epithelium [62].
Control of the activation and time span of gene expression can
be obtained by the use of inducible promoters that are triggered
by the presence of an additional agent that is released
endogenously under certain physiological conditions (e.g.,
ischemia) or is administered exogenously [209]. In corneal gene
therapy an E-selectin, tumor necrosis factor-inducible, promoter
was studied ex vivo, since tumor necrosis factor appears in
aqueous humor during corneal graft rejection. Expression of
chloramphenicol acetyl transferase was conditional on the presence of tumor necrosis factor in the experimental media [185].
There is often a need to identify an optimal DNA dose, since
increasing doses have shown a direct correlation with the magnitude of gene expression or knock-down, and also with pharmacological effect and side effects. For example, the percentage of
corneal surface area of lacZ gene expression following intrastromal
injection of naked DNA increased from 0.6% ±0.1% to 40.5% ±
7.5% as doses increased from 0.05 ng to 1800 ng, respectively
[16]. Elevated doses (1–4 μg) of shRNA plasmids complexed
with cationic liposomes were administered intrastromally to target
matrix metalloproteinase-9. This treatment enhanced the
corresponding gene knock-down in the murine cornea [142].
Higher doses (5–100 μg) of naked DNA encoding IFN-α1 were
administered topically to scarified corneas, resulting in increased
survival of mice following HSV-1 challenge [143]. Monkeys
injected intracamerally (i.e., to the anterior chamber) with high
(109 plaque-forming units) doses of adenovirus encoding green
fluorescent protein developed strong inflammatory reactions,
which were not observed at lower doses [17]. A connection
between increased adenovirus dose and the appearance of side
effects was also reported by Larkin et al. [119]. These examples
clearly illustrate the importance of optimizing the DNA dose.
Finally, the issue of toxicity is related to the design of corneal
gene delivery systems in that gene vectors substantially vary in
their safety profile, and as expected, some of them are not devoid
of corneal side effects. It is important to note that in the eye even
a mild inflammatory response might impair vision [21]. When
used in the cornea, intrastromal injection of naked DNA [16] and
both topical and intracameral administrations of lipoplexes [164]
have yielded no side effects. Viral vectors are generally more
toxic than non-viral vectors [210]. In the cornea, 3 studies involving intracameral injections [1,3] or topical administration
[14] of adenovirus were unable to detect cytopathic effects.
Ex vivo transduction of corneal graft using adenovirus has shown
that postoperative inflammation was not exacerbated by the
administration of adenovirus [18].
However, a few studies did exhibit mild to severe inflammation following intracameral administration of adenovirus
[17,120]. In one such study by Borras et al., the inflammatory
response that appeared in histologic examination demonstrated
no gross external manifestations such as redness or irregular
tearing [12]. This highlights the importance of awareness of
potential side effects and cytotoxicity in the design and evaluation of corneal gene delivery systems.
4.2. Modes of administration of corneal gene delivery systems
The mode of administration of corneal gene delivery systems
has a considerable impact on the study outcomes and applicability. When conducting research in corneal gene delivery,
the first decision that should be made regarding the mode of
administration is whether a systemic or an ocular mode of
administration is preferred. In the latter case, a choice can be
made between topical and invasive administration. Among the
invasive modes of administration, there are several alternatives
for the site of injection. Furthermore, there are additional invasive procedures that were used in preclinical studies to
enhance corneal gene transfer.
When comparing systemic to ocular modes of gene administration, it is clear that the latter carries the inherent advantages
of local delivery i.e., the ability to achieve enhanced local
expression levels and therapeutic effects combined with a lower
probability of systemic adverse effects [211]. Thus, numerous
studies involving corneal gene delivery, as seen in Tables 1–5,
have used transfer to ocular tissues such as the anterior chamber,
the vitreous body and the surface of the eye i.e., the cornea and
conjunctiva.
However, there are a number of studies that have employed
non-ocular gene administration to correct corneal diseases.
Most studies using nasal [116,138,144], but not vaginal [144] or
intramuscular [88,138], administration to prevent HSK were
successful, although to a lesser extent than ocular administration
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[138]. Systemic gene therapy that treated corneal manifestations
of mucopolysaccharidosis VII was successful in neonate mice
[114] and neonate dogs [112,113]. However, no pathological
correction was observed in adult mice, possibly due to the
blood-retinal barrier preventing transduction of adenovirus and
uptake of the transgene, β-glucoronidase. It was suggested that
immaturity of the blood-retinal barrier in the neonates may have
enabled the ocular adenoviral vector transduction [114].
Several studies describe the unequivocal success of corneal
gene delivery following peripheral administration. Sakamoto
et al. describe intramuscular administration of adenovirus
encoding soluble transforming growth factor β receptor to treat
corneal opacity and neovascularization. This treatment resulted in
elevated transgene levels in the supernatant of the homogenized
eye, similar to systemic gene expression [99]. Pardridge et al.
identified lacZ transgene expression in the cornea following
intravenous injection of pegylated immunoliposomes targeting
the mouse retina [115]. Ghiasi et al. succeeded in immunizing
mice against corneal HSV-1 infection using intramuscular
injections (primary and 2 boosters) of naked DNA encoding a
cocktail of 5 of the viral glycoproteins [131]. Larkin et al. and
Ritter et al. noted a substantial prolongation of corneal graft
survival following systemic administration of adenovirus encoding CTLA4-Ig or viral IL-10, respectively, in comparison to ex
vivo transduction of corneas prior to transplantation [79,119].
A study by Rouse et al. emphasized the advantages of the
ocular over the systemic mode of administration. An approach of
VEGF RNAi was used to study the suppression of HSK lesions
and corneal neovascularization by comparing subconjunctival
with intravenous injection. In order to increase the effectiveness
of the intravenous injection of the siRNA duplexes, which were
used in the subconjunctival injection, TargeTran™ was compounded with the RNAi duplexes and the dose was increased
4-fold. Combining these means the systemic administration
achieved an equivalent anti-angiogenic effect and a HSK clinical
score as local administration [146].
A major challenge for the ocular mode of administration is to
obtain substantial gene expression by topical administration, the
preferred ocular mode of administration for therapeutic agents
[176,212]. After application, the hydrodynamics of blinking
and tear flow immediately remove the delivery system from the
corneal surface [130,176]. It was also suggested that the tight
junctions of the epithelium and Bowman's membrane, might act
as a barrier for penetration of the adenovirus [102].
Among the studies that evaluated topical administration to
the naïve eye, in contrast to the scarified eye, only those studies
from 1996 by Matsuo et al. using a low dose (0.8μg) of cationic
lipoplexes [163,164] achieved corneal transgene expression.
Other studies using cationic lipoplexes [176], adenovirus
[14,102] and naked DNA [88,137,163] reported no detectable
reporter gene expression. One study exemplified the usefulness
of eye-drops in treating a disease model of unscarified, naïve
cornea. Application of naked DNA to the conjunctival sac has
yielded partial immunization against HSV-1, consequently
inhibiting stromal, but not epithelial, keratitis [130]. However,
it is important to note that even very low transgene expression
can yield successful immunization [208].
125
Methods were examined to augment corneal gene expression
following topical administration. One way to accomplish this is
to change the delivery system. For instance where naked DNA
has yielded negative results, cationic lipoplexes successfully
yielded expression of the lacZ gene [163]. An alternative,
which was used in a majority of studies involving HSK, is to
scarify the cornea. This procedure was shown to enhance and
improve the consistency of gene expression [88], although Carr
et al. noted that it has only limited relevancy for the clinical
setting [143]. Studies that have found no transgene expression
following topical instillation, and continued experiments using
invasive ocular administration i.e., intracameral [102] or
intravitreal [176] injections, have resulted in substantial
expression. The limited success of topical administration to
the naïve eye may explain the propensity to use invasive modes
of administration in preclinical studies. These include intrastromal, intralimbal, subconjunctival, intracameral and intravitreal injections. It should be mentioned that repeated
subconjunctival and intravitreal injections of drugs to patients
were associated with morbidity e.g., vitreous hemorrhages,
endophthalmitis and cataract [205,212].
The ocular mode of administration is correlated with the
intracorneal location of gene expression since the site of microinjection controls the type of cells that receive the transgene
[179]. Therefore, as exemplified from various studies (see
Table 1), intracameral and intravitreal injections yielded gene
expression in the endothelium, and intrastromal injections produced gene expression in the stroma, and in some cases in the
epithelium. Studies outlying this pattern of gene expression
describe lipoplexes administered intracamerally or intravitreally
yielding transgene expression in the epithelium, and in the
former case also in the endothelium [164]; and adenovirus
administered intracamerally yielding transgene expression in
both endothelium and epithelium [179]. The specific intracorneal layer where the gene was expressed was unrelated to the
vector type or animal species used.
From the reports using physical techniques to transfer
reporter genes to the cornea it is apparent that electroporation
[5,21,171] had no effect on the location of expression i.e., the
gene was expressed according to the injection site, and that
gene gun delivered the transgene to the epithelium [61,62]. It
was suggested that gene gun technology is inappropriate to
transfect stromal keratocytes due to their dense collagenous
matrix [62].
Additional means of modifying the mode of administration
to enhance corneal gene transfer in preclinical studies include
using invasive procedures for improving the accessibility of the
delivery system to the stroma. For example, Perez et al. created
a small tunnel from the corneal epithelium to the anterior stroma
using a 33-gauge needle [175]. Mohan et al. produced a corneal
flap to expose the corneal bed prior to instillation of the viral
solution [174]. Kitajima et al. created a stromal pocket to
implant a hydrated hydrogel that has a size of 1 × 2 mm and a
thickness of 0.2 mm, and was pre-incubated with antisense or
sense oligonucleotides [181]. Okuyama et al. formed a lamellar
keratotomy using a microscalpel and subsequently inserted a
sponge soaked with viral solution into the wound for a few
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minutes. Lamellar keratotomy does not usually cause scars that
impair vision. In fact, lamellar keratoplasty, a more invasive
procedure, is executed repeatedly in the clinic [102].
Sakamoto et al. highlight the importance of not interfering
with the transparency of the central cornea [6,171] as under this
condition it is possible to maintain good vision even if the
peripheral cornea is hazy [21]. Challa et al. report that the specific
technique used for intracameral injection determines whether
corneal epithelial neovascularization will develop, presumably
due to endothelial damage [177]. These issues pertain to various
modes of administration.
4.3. Animals used in corneal gene delivery studies
Mice are by far the most commonly used animal species in
experimental gene therapy, in general [213], and their use is
also more common than other species in experimental corneal
gene therapy, see Fig. 3A. The animal species and model
chosen for a preclinical study affect its chances of success
and relevancy. The ultimate decision regarding the choice of
animal is a compromise between factors, which vary in
their importance for research of different corneal diseases. To
assist in this decision for corneal gene therapy, the following
considerations can be taken into account, and some examples
follow:
4.3.1. Inter-species variability in the anatomy, physiology and
immunology of the eye
Examples include eye size, immunological response and the
ability of the corneal endothelium to replicate. The fact that the
rabbit eye is similar in size to the human eye was mentioned as
an advantage in using the rabbit to study corneal gene delivery
[12]. In contrast, the small size of the murine eye was pointed
out as a disadvantage in efforts to follow clinical signs of ocular
disease progression. Topical immunological challenge in rats is
difficult due to the unusual structure of their conjunctiva [214].
The corneal endothelium is mitotic in rodents [18,75] while in
rabbits it is potentially mitotic [23]. However, since its
replicative capacity is low [185,192] some researchers consider
it to be virtually amitotic [215]. The corneal endothelium is
amitotic in humans [18,23], sheep [18], cats [18,23] and
monkeys [23]. This is particularly germane to corneal gene
therapy studies where the endothelium plays a significant role
e.g., corneal graft rejection.
4.3.2. The existence of an animal strain that mimics human
pathology
Mucopolysaccharidosis VII mice [102,112], cats [112] and
dogs [112,113] develop a disease that is similar to human
mucopolysaccharidosis VII in many features. Such mice and
dogs were used in gene therapy studies that also evaluated the
effect of the treatment on the cornea [102,112–114,162].
Fig. 3. (A) The animal species used for corneal gene transfer studies;
(B) according to the application of delivery, where corneal haze includes
opacity, wound healing and corneal manifestations of mucopolysaccharidosis
VII, during 1994–2006. Data were compiled from studies mentioned in Tables
1–5, studies involving extra-ocular modes of administration [99,112–116] and
additional studies [107,109–111]. In the preparation of B, for studies that
included both a reporter and a therapeutic gene, only the therapeutic gene was
taken into account. For studies that used correction of neovascularization for
either treatment of HSK or prevention of graft rejection, the neovascularization
part of the study was not taken into account.
4.3.3. The disease pattern and symptoms in the animal model
in comparison to humans
The fact that HSK lesions in mice following primary
infection mimic the human disease was noted by Rouse et al.
as the primary reason for the popularity of murine model in
preclinical studies of HSK [216]. The process of corneal graft
rejection in sheep is similar to that in humans [217]. In mice and
rats, unlike humans, the process is acute [75] and rejection lines
in rats are infrequent. In rabbits the process is slow and there is
the need to accelerate graft rejection by induction of pregraft
neovascularization and inflammation [217]. It was suggested
that this procedure changes the disease progression from a
model of low-risk to high-risk transplantation in humans [75]. A
different example is a model of corneal haze in rabbits where the
symptoms peak 2 to 3 weeks post-treatment, which is significantly faster than in humans [159].
4.3.4. Past experience in corneal gene delivery studies of a
connection between managing a specific disorder
and the use of a particular animal species
When relating the disease treated to the animal species
utilized it is apparent that almost all corneal gene therapy
studies treating HSK have used mice, see Fig. 3B.
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
4.3.5. The availability of analytical methods, including investigational reagents, to conduct the study in a particular animal species
The fact that there are various immunological reagents
available for scientific work with mice [214] has contributed to
use of this animal species in transplantation [217] and in HSV-1
[145] studies. There are also cytokine probes and cell-surface
markers available for sheep, presumably due to their economic
importance [217].
4.3.6. Past experience in husbandry of the animals under study
in research facilities
Williams et al. introduced the use of sheep for corneal
transplantation studies and highlighted technical aspects in their
husbandry and use e.g., caging each animal in the company of
at least one other animal and installing mirrors at eye level to
raise the apparent flock size [217]. Despite such practical
suggestions, the fact that sheep are not commonly housed in
research institutions might require additional preparative work
in their husbandry as a part of a corneal gene delivery study.
4.3.7. Modifications and improvements of existing animal
models that are suggested in contemporary publications
Perng et al. described a high-reactivation HSV-1 strain that
following infection of the rabbit cornea causes scarring on days
35–58 post-infection in the majority of animals. Since the
animals were free of scarring on day 30, the authors suggested
that this model mimics recurrent episodes of human HSK that
include neovascularization and scarring. HSV-1 strains with
low-reactivation phenotypes yielded significantly less scarring
[218]. It should be noted that while murine models involving
reactivation of HSV-1 have been reported, the most common
murine model for HSK involves a primary infection [216].
The importance of choosing an optimal animal model for
corneal gene therapy was highlighted in a discussion in the
United States Department of Health and Human Services,
National Institutes of Health regarding an application to use a
retroviral vector in a clinical study to treat superficial corneal
opacity and scarring. A concern that addressed the preliminary
studies, which aimed at supporting the application, was
“whether higher rates of haze in the rabbit make it an inadequate
animal model of the condition” [219].
5. Conclusions and future perspectives
Along with tissue engineering [220,221], gene therapy
approaches stand on the front line of advanced biomedical
research to treat blindness arising from corneal diseases, which
are second only to cataract as the leading cause of vision loss
[220]. The significance and prevalence of corneal diseases
were the driving force behind the studies in corneal gene
therapy that have created a rich body of evidence in the
literature, which can guide future studies and opportunities in
corneal gene therapy.
Topical delivery to the eye is the most convenient way of
ocular gene delivery. However, the challenge of obtaining
substantial gene expression following topical administration has
127
led to the prevalence of invasive ocular administration. The
design of advanced delivery systems that prolong the contact time
of the vector with the surface of the eye may enhance transgene
expression, thereby enabling non-invasive administration.
Corneal gene delivery studies have shown that corneal gene
expression can be obtained by ocular administration, most
commonly invasive by its nature. Intracameral and intrastromal
administrations have generally yielded expression in the corneal
endothelium and the stroma (and sometimes also in the
epithelium), respectively, regardless of the vector type or
animal species studied. This means that when designing corneal
gene transfer studies, the mode of administration should be
linked to the corneal layer where gene expression is desirable,
which in turn depends upon the disease being treated.
Viral vectors carry the risk of eliciting pronounced immune
responses although their use is known to yield stronger gene
expression that expands over a longer time span, when compared
to use of non-viral vectors. All of these traits were exemplified in
corneal gene therapy studies and should be taken into account in
future studies. Moreover, some of the new vectors that are constantly being designed are (a) safer; (b) have the potential to
improve the gene transfer process, thereby achieving an enhanced effect, and (c) allow control over activation and deactivation of gene expression by the use of inducible promoters.
For example, episomal plasmids that replicate during the cell
cycle and segregate into daughter cells, prolong the transgene
expression in comparison to traditional plasmids [222]. Highcapacity adenovirus not only extends the transgene expression
but is also safer than previously available adenoviral vectors
[223]. The repertoire of pharmacologically-induced promoters
that are triggered by clinically available agents was expanded
[224]. Implementation of new vectors in corneal gene delivery
studies is expected to yield valuable results.
Corneal gene therapy studies yielded positive outcomes in
combating major acquired corneal disorders such as corneal graft
rejection, neovascularization, haze and HSK. Studies were also
conducted to correct ocular manifestations of an inherited
metabolic disease, mucopolysaccharidosis VII. One way of
advancing the research in this field is to manage corneal disorders
or corneal manifestations of metabolic diseases that have not yet
been studied with gene transfer approaches. In particular,
inherited corneal diseases are relevant candidates for corneal
gene therapy.
A choice of an appropriate animal species and model is
important to increase the chances of success and usefulness of
the study. A way to enhance the relevancy of preclinical studies
in gene therapy that aim to treat inherited disorders, is to utilize
animal strains that mimic the human inherited disease as closely
as possible. This was conducted in several studies involving
mice and dogs that develop mucopolysaccharidosis VII. Genetic
engineering of animals having inherited corneal diseases that are
similar to human corneal disorders will advance the field and
facilitate expansion of the array of diseases that are, and
eventually can be, studied by corneal gene transfer.
Clearly, significant progress has been accomplished in
the field of corneal gene therapy. A challenge remains to
characterize the kinetics of corneal gene expression with the
128
E.A. Klausner et al. / Journal of Controlled Release 124 (2007) 107–133
emphasis on understanding the barriers and rate limiting steps
underlying the gene transfer processes. Information about the
nature of the interaction between corneal cells and the gene
vector will assist in the design of optimized delivery systems.
This will facilitate the advance from preclinical to clinical
studies, which have not yet been conducted for corneal gene
therapy. The goal of translating corneal gene delivery systems to
pharmaceutical products is yet to be achieved.
Acknowledgement
Eytan Klausner, Robert Chapman and Shridhar Andurkar
thank Midwestern University Chicago College of Pharmacy for
supporting this work.
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Glossary
CMV; cytomegalovirus
CTLA4-Ig; cytotoxic T lymphocyte antigen 4-immunoglobulin
gD; HSV-1 glycoprotein D
HSK; herpetic stromal keratitis
HSV; herpes simplex virus
IFN; interferon
IL; interleukin
MIDGE; minimalistic immunologically defined gene expression
RNAi; RNA interference
shRNA; short hairpin RNA
Th1; T-helper 1
Th2; T-helper 2
VEGF; vascular endothelial growth factor