Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

found in the eye (and other tissues) and is one of the strongest anti-angiogenic compounds known (Dawson et al., 1999). Because these factors are secreted, therapy can be achieved by targeting cells distant from the site of disease. Therapeutic protein secreted by these cells can then diffuse to the lesion. Another successful strategy has been to deliver a soluble decoy receptor to VEGF (s-Flt) which binds excess VEGF preventing pathologic neovascularization (Lai et al., 2002b).

1. Corneal neovascularization

Specific studies that have been performed in corneal neovascularization models have included vectoral delivery of endostatin, antisense VEGF RNA, brainspecific angiogenesis inhibitor 1, and kringle 5 of plasminogen. More recently, topical delivery of siRNAs directed against VEGF or its receptors has also been shown to suppress corneal neovascularization (Lai et al., 2002a; Yoon et al., 2005; Yu et al., 2003; Zhang et al., 2003a).

2. Choroidal neovascularization (CNV)

Viral vector-mediated delivery of both anti-angiogenic proteins and molecules that inhibit the endogenous pro-angiogenic factors have successfully diminished the pathology of choroidal neovascularization in rodent and non-human primate (NHP) models (Auricchio et al., 2002; Reich et al., 2003; Tolentino et al., 2004). Campochiaro et al. (2006) conducted a Phase I gene therapy clinical trial in individuals with advanced neovascular AMD. This was a dose escalation study using a serotype 5 (Ad5), E1-, partial E3-, E4-deleted adenoviral vector carrying human pigment epitheliumderived factor (PEDF) injected into the vitreous of 28 subjects. The results from this trial were encouraging, in that there were no serious adverse events or dose-limiting toxicities. Although the study was designed to assess safety and not efficacy, there was

evidence that the high dose treatments resulted in anti-angiogenic activity lasting several months.

3. Age-related macular degeneration (AMD)

To date, many of the gene therapy studies for AMD have focused on one of the major complications of this disease – choroidal neovascularization (see above). Molecular genetic studies will continue to identify genes and molecular pathways that could become additional targets for AMD gene therapy. The ATP-binding cassette transporter gene (ABCR) appears to play a significant role in the pathogenesis of AMD (Allikmets, 2000). More recently, genes involved in immunologic pathways have been implicated in AMD pathogenesis. In particular, Complement Factor H (CFH) has emerged as a particularly interesting molecule in the search for genebased treatments of this disease. CFH and in particular the Y402H variant were identified to predispose to AMD simultaneously by four different groups (Edwards et al., 2005; Hageman et al., 2005; Haines et al., 2005; Klein et al., 2005). More than three dozen additional papers have been published on this association and these findings are likely to result in gene-based treatment strategies.

4. Diabetic retinopathy

Diabetic retinopathy is a microvascular complication in the retina resulting from diabetes mellitus. To date, gene-based treatment strategies have targeted a late pathological feature – inner retinal neovascularization (see above). There are additional possible genetic targets for future studies in diverse biochemical pathways. Such pathways include oxidation of retinal cells, polyol accumulation pathways, sphingolipid metabolism, insulin receptor signaling, non-enzymatic glycation in retinal cells and the release of growth factors by endothelial cells (Bronson et al., 2003).

E. IOP Lowering Strategies

1. Glaucoma

Elevated intraocular pressure (IOP) is one of the main risk factors for glaucoma, a group of optic neuropathies, and lowering IOP remains the main therapeutic approach in the treatment of glaucoma. Most of the current pharmacological approaches to lowering elevated IOP have been developed to decrease the production of aqueous humor or improve uveoscleral outflow instead of specifically targeting the TM to enhance conventional outflow. It is known that the conventional pathway accounts for 50% to 75% of aqueous outflow (Gupta, 2005), and the resistance to outflow primarily resides in the juxtacanalicular region of the TM and the inner wall of Schlemm’s canal (SC). Increasing knowledge of TM physiology and molecular biology has shown that this tissue has unique morphologic and functional properties involved in the regulation of aqueous humor outflow. Gene therapy-based studies are being developed to target the structures and enzymes involved in maintaining cell shape, cell–cell and cell–extracellular matrix (ECM) interactions that could ultimately influence cellular and tissue contractility/relaxation, in these areas. The actin cytoskeleton, which mediates a variety of biological properties in cells, including cellular contractility and adhesions, has become a target for IOP lowering gene therapy strategies.

Over the last decade, compounds capable of disturbing cellular contractility have been demonstrated to lower IOP and increase outflow facility in organ cultured ocular anterior segments (Khurana et al., 2003; Song et al., 2005b) and in rats and primates in vivo (Peterson et al., 1999; Tian et al., 1998). Similar results have been accomplished by overexpression of proteins that modulate contractility and consequently reduce resistance to aqueous humor outflow and increase outflow facility. Rho GTPase (Rho), which in its active GTP-bound

form interacts with specific effectors to regulate actin and to mediate a variety of cell functions, could be a suitable target for gene therapy to lower IOP. Inhibition of Rho kinase, a critical downstream effector of Rho, has been shown to increase outflow facility in organ cultured porcine and human eyes, and in rabbit and monkey eyes in vivo (Honjo et al., 2001; Rao et al., 2001; Tian and Kaufman, 2005). Interference with other proteins associated with Rho’s cytoskeleton regulating cascade include myosin II ATPase (Zhang and Rao, 2005), protein kinase C (Khurana et al., 2003; Tian et al., 2000). G-protein coupled receptors (Mettu et al., 2004; Rao et al., 2005b) have also been shown to alter outflow facility via the conventional outflow pathway.

It is reasonable to hypothesize that an increase in outflow facility could be achieved using gene therapy strategies that express genes encoding protein inhibitors of this Rho associated cascade, or by expressing dominant negative mutants of the actin effectors. An increase in outflow facility was observed in organ cultured human eye anterior segments overexpressing dominant negative Rho GTPase, the upstream regulator of Rho-kinase (Vittitow et al., 2002). Rao et al. (2005a) showed that dominant negative Rho kinase (DNRK) increased outflow facility via the TM in organ cultured human eye anterior segments, and correlated these findings with the status of myosin light chain (MLC) phosphorylation, cell shape, and actin cytoskeletal organization in human TM cells in vitro. However, the percent increase in facility with DNRK (80%) was much greater than that reported with dominant negative Rho GTPase (30%). Both of these observations underscore the potential importance of the Rho/Rho-kinase pathway in the regulation of aqueous humor outflow through the TM, and demonstrate that selective inactivation of Rho-kinase can lead to increased outflow facility (Tian and Kaufman, 2005). This difference between the outflow facility effects of inhibiting Rho compared to Rho-kinase suggests that

direct targeting of Rho-kinase represents a more effective strategy for modulating aqueous outflow.

The exoenzyme C3 transferase (C3) from Clostridium botulinum specifically inactivates Rho by ADP-ribosylation (Aktories et al., 1992). Ad-mediated C3 delivery significantly disrupted the cytoskeleton, focal adhesions and cell–cell adhesions of cultured human TM (HTM) cells, and increased outflow facility by 90% in organ cultured monkey anterior segments (Liu et al., 2005).

Both the smooth muscle and nonmuscle isoforms of the actin binding protein caldesmon are potent inhibitors of actin– tropomyosin activated myosin MgATPase. The non-muscle isoform is a regulatory factor in the microfilament network in cells, controlling the assembly and stabilization of microfilaments (Huber, 1997). The overexpression of non-muscle caldesmon induced changes in the actin cytoskeleton distribution, resulting in the formation of unique curvy actin networks and the disruption of focal adhesions in cultured HTM cells (Grosheva et al., 2006). It also increased outflow facility in organ cultured human and monkey anterior segments (Gabelt et al., 2006).

Other gene therapy strategies with potential to lower IOP include regulating matrix proteins in both conventional and uveoscleral pathways, overexpressing prostaglandin (PG) synthase 2 in the ciliary body and decreasing aqueous humor production. The ability to deliver genes to related cells/tissues such as the TM, cilary muscle (Kee et al., 2001) and non-pigmented ciliary epithelial cells (NPE) (Liu et al., 1999) has been demonstrated. RNA interference-based gene therapeutic strategies have been developed targeting inhibition of aqueous humor formation. Topical administration of specific siRNAs targeting carbonic anhydrase genes (Pintor et al., 2006) and alpha and beta adrenoceptors (Jimnez et al., 2006) reduced IOP in rabbits. The reduction of IOP with these strategies is comparable to that produced by commercial products. Moreover,

siRNA (and particularly vectored delivery of siRNA) has the advantage of potentially producing a long lasting effect compared to commercial pharmaceutical products (Jimnez et al., 2006; Pintor et al., 2006). Overexpressing enzymes in PG biosynthasis (e.g. PG synthese 2/cyclooxygenase-2 (COX- 2)) in the ciliary muscle could be another approach for lowering IOP by enhancing uveoscleral outflow. FIV-mediated COX-2 reduced IOP but did not decrease aqueous humor formation in live cats (Barraza et al., 2007). This is suggestive of an effect on uveoscleral outflow.

F. Neuro-Protection/Rescue Strategies

Selective demise of RGCs is the hallmark of virtually all diseases of the optic nerve, including glaucomatous optic neuropathy. Similarly, apoptotic death of photoreceptors occurs in all inherited forms of RP. Strategies for blocking apoptotic cell death in RGCs and photoreceptors include: altering the balance of proand anti-apoptotic molecules, delivering inhibitors of apoptosis, and delivering neurotrophic factors which delay apoptotic cell death.

1. BCL2 activation/BAX inhibition

Neuroprotection has been demonstrated following expression of genes or their protein products which interfere with the apoptosis cascade. Martinou et al. (1994) reported a transgenic mouse line expressing Bcl-2, an apoptosis inhibitor that protects the integrity of the mitochondrial membrane, in rat neurons. A 50% increase in retinal ganglion cell numbers, accompanied by an increase in the thickness of the inner plexiform layer, was noticed in comparison to normal mice. Malik et al. (2005) showed that RGC cells transduced with AAV vector expressing Bcl- 2 remained morphologically intact and survived up to 8 weeks after axotomy. Bennett et al. (1998) delivered Bcl-2 to degenerating photoreceptors of a mouse model of retinitis pigmentosa and showed delayed degeneration of these cells.

2. Caspase inhibitors

Central to the apoptosis cascade is the activation of cysteine proteases, termed caspases. Ocular delivery of genes coding caspase inhibitors might be a useful strategy to protect RGCs from glaucomatous damage. McKinnon et al. (2002) showed that AAV vector-mediated expression of human baculoviral IAP repeat-containing protein-4 (BIRC4), a potent caspase inhibitor, promoted optic nerve axon survival in a rat model of experimental glaucoma. This was supported by the observation that inhibition of retinal caspase-3 activity with BIRC4 reduced caspase-3-mediated cleavage of alpha-fodrin, a neuronal cytoskeletal protein and a known caspase-3 target (Tahzib et al., 2004).

3. X-linked inhibitor of apoptosis (XIAP)

Renwick et al. (2006) recently reported successful AAV-mediated XIAP gene therapy in rat eyes with elevated IOP-induced ischemic damage to retinal neurons. Functional analysis revealed that XIAPonly treated eyes retained larger b-wave amplitudes than GFP-only treated eyes up to 4 weeks post-ischemia. The number of cells in the inner nuclear layer (INL) and the thickness of the inner retina were significantly preserved in XIAP-treated eyes compared to GFP-treated eyes.

4. Neurotrophic factors

Several studies have shown that apoptotic death of RGC after axonal injury can be prevented in the short term in animal models by repeated intravitreal injection of neurotrophins such as neurotrophin-4 (NT-4) (Peinado-Ramon et al., 1996), BDNF and CNTF, and sciatic nerve (ScN)-derived medium (Klöcker et al., 1997; Mey and Thanos, 1993; Peinado-Ramon et al., 1996). However, these procedures have some disadvantages including side effects induced by multiple intraocular injections and short half-lives of the proteins. Recently,

encouraging results with intraocular delivery of the corresponding genes have been reported (Martinou et al., 1994; Renwick et al., 2006; Vittitow et al., 2004). These genes also slow retinal degeneration in animal models of RP.

a. Brain-derived neurotrophic factor (BDNF) –

BDNF is an important survival factor for RGCs. Ad-mediated intravitreal delivery of BDNF selectively transduced Müller cells and the expressed BDNF from these cells protected RGCs in a rat optic nerve transection model (Di Polo et al., 1998). Admediated delivery of BDNF to Müller cells has also resulted in preservation of structure and function of light-damaged photoreceptors, a model of macular degeneration (Gauthier et al., 2005).

Electroporation of GDNF into rat ganglion cells led to short-term rescue of axotomized RGCs (Ishikawa et al., 2005). AAV has also been used to deliver BDNF to the retinas of animal models of retinal disease. Following a single intravitreal injection of AAV, a highly efficient transduction of RGCs was achieved in a rat model of glaucoma. In this model it was found that AAVmediated gene therapy with BDNF has a significant neuroprotective effect compared to saline or control virus injections (Martin and Quigley, 2004). BDNF receptor TrkB is markedly downregulated after axotomy of the optic nerve. AAV-mediated TrkB gene transfer into RGCs combined with exogenous BDNF administration markedly increased neuronal survival (Cheng et al., 2002).

Recent studies indicate that neuroprotection afforded by BDNF is mediated by extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K). The role of the Erk1/2 pathway in the survival and axon regeneration of adult rat RGCs has also been studied. Pernet et al. (2005) used AAV to transduce axotomized rat RGCs in vivo and identified that the Erk1/2 pathway plays a key role in the protection of RGCs. Their group also used a

vector with genes encoding constitutively active or wild-type mitogen-activated protein kinase 1 (MEK1), the upstream activator of Erk1/2. MEK1 gene transfer into RGCs markedly increased survival, indicating that the Erk1/2 pathway also plays a key role in the protection of RGCs from ocular hypertensive damage (Zhou et al., 2005).

b.Ciliary neurotrophic factor (CNTF) –

Ad-mediated expression of CNTF led to better preservation of intraretinal RGCs, but did not support regeneration of axotomized RGCs into peripheral nerve grafts (Bok et al., 2002; Schlichtenbrede et al., 2003; Weise et al., 2000). Ad-mediated delivery of CNTF has also been used to slow photoreceptor degeneration in RCS rats (Huang et al., 2004).

AAV-mediated delivery of CNTF has resulted in much more impressive rescue of photoreceptors in animal models of retinitis pigmentosa (Liang et al., 2001b). Unfortunately, at the doses used, there was also some evidence of impairment in visual function (Bok et al., 2002; Liang et al., 2001a; Schlichtenbrede et al., 2003). Such findings were not reported after delivery of CNTF via encapsulated cells (see above).

c.Glial cell line-derived neurotrophic factor (GDNF) – AAV-mediated delivery of GDNF prolonged photoreceptor survival in rodent models of retinal degeneration, including

atransgenic mutant rhodopsin rat model of RP (McGee Sanftner et al., 2001). When AAV-GDNF was co-delivered with a vector designed to deliver a wild-type version of the defective gene in two additional animal models with loss-of-function disease, photoreceptor survival was further enhanced

over gene augmentation therapy alone. The two animal models were the Prph2Rd2/Rd2 mouse (defective in Rds/Peripherin, also known as Prph2) and the Royal College of Surgeons (RCS) rat (defective in Mertk) (Buch et al., 2006).

d.Pigmentepithelium-derivedfactor(PEDF)–

PEDF is unique in that it has both neurotrophic and anti-neovascular properties. Results of delivering PEDF in animal models of ocular neovascularization are discussed above. Ad-mediated delivery of PEDF reduced ischemic injury and photoreceptor apoptotic death in a rat model (Takita et al., 2003). Lentivirus-mediated delivery of PEDF also protected against retinal degeneration in the RCS rat (Miyazaki et al., 2003).

e.Fibroblast growth factor 2 (FGF2) –

FGF2 exhibits mitogenic, angiogenic, wound healing, and neuroprotective properties. In animal models with retinal degeneration, intravitreal injection of recombinant FGF2 significantly delayed

photoreceptor cell death (Faktorovich et al., 1992; Lin et al., 1997)). Spencer et al. tested the feasibility of HSV-mediated FGF2 for neuroprotection both in vitro and in vivo. HSV-mediated FGF2 expression prolonged survival of PC12 cells in culture, promoted differentiation of these cells to the neuronal phenotype, and resulted in protection against photoreceptor loss in rats with light induced photoreceptor damage (Spencer et al., 2001). AAV-mediated FGF2 delivery also showed neuroprotective effects in animal models with retinal degeneration (Sapieha et al., 2003, Schuettauf et al., 2004).

G. Anti-Tumor Strategies

Hurwitz and colleagues (Chevez-Barrios et al., 2005) delivered an Ad vector carrying a herpes simplex virus thymidine kinase gene (AdV-TK) directly into vitreous tumor sites in 8 different subjects who had failed therapies for retinoblastoma. This was a Phase I (safety) trial. Each injection was followed by an intravenous injection of ganciclovir, which kills the TK-expressing cells. Similar to the Campochiaro et al. study, there was no serious toxicity. The 7 patients who were treated with doses 1010

vector particles had resolution of their vitreous tumor. Some of the therapeutic effect could have been related to a “bystander effect”. One patient even remained free of active vitreous tumor 38 months after treatment (Chevez-Barrios et al., 2005). These results provide support for going forward with a Phase II (efficacy) trial.

IV. CONSTRAINTS AND

CHALLENGES

The development of gene therapy as a novel therapeutic approach to ocular disease will depend on continued identification and characterization of the genes, gene products, developmental regulatory signals, and pathways involved in the disease processes. Much work still needs to be done on vector and delivery method refinements, gene mapping, animal models, and gene expression profiling, all of which will play important roles in altering the neurodegenerative process.

A. Gene-Targeting Technology

Gene expression profile and proteomic analyses of the TM and of other cells/ tissues involved in ocular disease have led to the establishment of published libraries. These libraries provide important information about the genes and proteins preferentially expressed, and will further the goal of directed gene delivery to the specific cell types that are most affected by disease. Gene-based therapies will require such cell-specific targeting and prolonged gene expression to be effective alternatives to current therapy. Each delivery system has advantages and disadvantages, and it is likely that no one vector is ideal for all strategies. The gene targeting tropism is one of the most challenging problems. Another challenge is presented by cargo limitations of the viral vector. AAV2/2, for example, is limited to a carrying capacity of 4.8kb, which could be a limitation for large cDNA

or regulatory elements. Other vectors can carry much larger transgene cassettes, including multiple genes and extensive regulatory sequences, but may be more difficult to produce, or may result in an unfavorable immune response. With continued progress in design and modification of viral vectors there will be more opportunities to transduce specific ocular cell targets with minimal inflammatory sequelae.

B. Tissue Specific Promoter

Even though intracameral delivery of vectors carries the viruses directly to the TM via aqueous humor flow, the use of TM-specific promoters should significantly increase the efficiency of gene transfer to TM without greatly affecting other ocular tissues.

Most gene therapy research in retinal disease (glaucoma and retinal degeneration) has used constitutive promoters such as the human cytomegalovirus (CMV) promoter or chicken beta actin (CBA) promoter. The CMV promoter can drive expression in multiple retinal cell types including Müller cells, vascular endothelial cells, rod photoreceptors, and other retinal neurons (Bennett et al., 1997b, 1999). Liu et al. (2002) compared promoter usage in primary differentiated and SV40 TAg transformed HTM cells (HTM and TM1 cells). In primary HTM cells, CMV was the only promoter displaying substantial activity. In TM1 cells, several promoters were functional with the order in decreasing activity being EF-1 alpha or CMV or UB6IE4/5. The results show the type of cell used is likely to be a crucial factor in evaluating the functions of promoter elements for genes expressed in the TM. Expression in transduced RGCs does occur with the CMV promoter, but in many studies, the proportion of RGCs transfected is relatively low or unquantifiable. Recently developed AAV vectors using the CBA promoter drive highly efficient transgene expression in RGCs, rod and cone photo-receptors, and

pigmented epithelium (Flannery et al., 1997; Mckinnon et al., 2002). Selective expression of transgenes in specific cell types can be achieved by driving the transgene with the appropriate promoter. For example, for rod photoreceptors, one can use the rhodopsin promoter (Bennett et al., 1998), for RPE, one can use the vitelliform macular dystrophy (VMD2) promoter (Esumi et al., 2004) and for ganglion cells, the synapsin-1 promoter (Morimoto et al., 2002).

Previous studies of gene expression profiles by single-pass sequencing of cDNA clones have provided some information about those genes that differentiate TM cells from other cell types. One such gene is the matrix Gla protein (MGP) (Gonzalez et al., 2000; Tomarev et al., 2003). Gonzalez et al. (2004) showed that infection in organ cultured human eyes with an Ad vector with an MGP promoter resulted in reporter gene expression in the cells of the conventional outflow pathway and did not show any activity in the corneal endothelium or other cells posterior to the scleral spur.

Liton and coworkers compared gene expression profiles between SC and TM primary cultures and identified several genes with promoters potentially capable of targeting gene expression to specific cells within the outflow pathway. The Ch3L1 promoter showed specific activity in expression of genes in a subset of TM cells in both cell culture and in perfused anterior segments. Results with the Ch3L1 promoter indicated that two different cell subtypes may be present in the TM. This study provides a potential new tool to investigate the role of these different cell types in both normal and pathophysiological function of the outflow pathway, with implications for possible future glaucoma gene therapy (Liton et al., 2005).

C. Gene Delivery to the Anterior and Posterior Segments

Gene therapy targets have been identified in both the anterior and posterior

segments of the eye. Both areas are accessible and easily visualized. Gene delivery is most commonly achieved through injection of viral-based vectors. Intravitreal, subretinal and intracameral injection methods are relatively safe and stable, and high levels of transduction have been achieved in targeted tissues via each approach. There are limitations as to how often injections can be made, especially intracameral injections, where repeated injections can lead to corneal changes or increased risk of infection (Borras et al., 2001).

The subretinal route of delivery leads to preferential expression in the RPE, making it especially useful for anti-angiogenic gene therapy for the treatment of ocular neovascular disorders. Expression is seen in photoreceptors with this route, given the appropriate capsid or envelope (Auricchio et al., 2001). The Rho (rhodopsin) promoter attached to an HIV vector increased both expression and photoreceptor cell specificity compared to a CMV promoter (Miyoshi et al., 1997). While repeated subretinal injection of AAV does result in a systemic immune response, cells are still transduced (Anand et al., 2000).

Intravitreal injections of AAV vectors have been shown to transduce RGCs (Leaver et al., 2006; Schuettauf et al., 2004) as well as tissues in the anterior segment (Martin et al., 2002). Ad can target Müller cells after intravitreal or subretinal injection, but the potential for a host immune response to Ad may be greater following intravitreal injection (Hoffman et al., 1997). Cells in the ciliary body and photoreceptors may also be transduced via Ad using this injection method (Von Seggern et al., 2003).

Biodistribution following posterior segment injection of AAV vectors has been investigated by several groups. Vector DNA was detected in the optic nerve following subretinal injection of rAAV-2/2, -2/4, or -2/5 in rats, dogs and monkeys and in the brain after intravitreal delivery of rAAV-2/2 in dogs and monkeys (Acland et al., 2005; Jacobson et al., 2006; Provost

et al., 2005). In another study, high levels of GFP were noted for at least 6 months in optic nerves and brains of mice and dogs after intravitreal delivery of rAAV–GFP (Bennett et al., 2000; Dudus et al., 1999). In both species, GFP was detected in the central nervous system (CNS) tissues known to be innervated by retinal ganglion cells. The authors concluded that toxicity studies of rAAV-mediated retinal gene therapy should assess the possibility that intraocular delivery of AAV can result in delivery of the transgene product from the retina to the brain. Intravitreal injection resulting in detection of gene products in the CNS was reported in a study in which recombinant AAV encoding β-glucuronidase was injected into the vitreous humor of young adult mice. β-Glucuronidase activity was subsequently detected in the brains of the recipients (Hennig et al., 2003).

Therapeutic strategies for glaucoma involving gene delivery to the anterior chamber primarily target the TM. Delivery of genes to the anterior chamber (Hernandez et al., 1999; Yang et al., 1994) via intracameral injection has been shown to effectively transduce the TM with Ad, HSV and LV vectors (Borras et al., 2002, 2006; Liu et al., 1999). The prostaglandin pathway gene COX-2 significantly decreased IOP with no reduction in aqueous humor formation, implicating an effect on the ciliary body and uveoscleral outflow pathway.

D. Long-Term Gene Expression

Glaucoma is a chronic condition; thus long-term expression of the transgene will be required for any gene therapy approach for this disease (Borras et al., 2002). Longterm (over 2 years) gene expression in the TM has not yet been reported. Liposome, Ad and HSV vectors have been limited by short duration, inflammation, or lack of sufficient targeted transduction. As described above, an LV vector was able to induce a relatively high level of reporter transgene expression in the TM of cat and

monkey models for at least 12 months after a single transcorneal injection (Loewen et al., 2004; Poeschla et al., 2006). Some AAV vectors appear to be able to mediate long-term gene expression in RGCs (Dudus et al., 1999).

E. Vector Associated Side Effects

One of the major disadvantages of viral vectors remains the induction of an inflammatory reaction. Severe inflammatory reactions, development of high neutralizing antibody titers and cell-mediated immune response have been reported with Ad (Bennett, 2003; Raper et al., 2003; Yang et al., 1994). Anterior chamber side effects associated with viral vectors include iritis, corneal edema, and keratic precipitates. With the FIV vector, strong GFP expression and a brief period of inflammation was associated with loss of expression in cats, rabbits and monkeys (Doi et al., 2004; Loewen et al., 2001). Anterior chamber inflammatory reactions (1–4 cells, trace to 3 flare) were seen in monkey eyes that received single, intracameral injections of varying doses of Ad and FIV. These were mostly resolved after several weeks. A transient fall in IOP was concurrently observed, with return to baseline levels as inflammation subsided (unpublished data). Immune responses to intraocular AAV are generally benign (Bennett, 2003; Reichel et al., 2001) although the majority of studies to date have used naive animals. Given the prevalence of seroconversion to AAV in the human population (Erles et al., 1999; Reichel et al., 2001), further studies regarding immunological limitations are warranted.

F. Repeated Injections

Though seemingly well tolerated via intravitreal or retinal injections, little data are available on repeated intracameral injections of viral vectors. One study has reported successful multiple injections in mice that received intravitreal or periocular Ad

(Hamilton et al., 2006). In monkeys, repeated intracameral injections with Ad caused persistent corneal edema and anterior chamber inflammation (Borras et al., 2001). A second intracameral injection of FIV/GFP construct resulted in no GFP expression and a prolonged period of mild inflammation in two monkey eyes (unpublished data). Subretinal readministration of an AAV vector resulted in additional transduction events despite significant serum antibody responses to the vector in monkeys (Bennett et al., 1999). While short-term immunosuppression was effective in blocking inflammation and subsequent rejection of the LV vector after subretinal injection in rabbits, the extensive drug regimen used may not be practical for many patients (Doi et al., 2004).

E. Restriction Factors

Recently, the restriction factor TRIM5α has been identified that blocks FIV replication. Currently the only known way to block the Lv1 restriction by monkey TRIM5α is to saturate it, which may be achieved by the addition of virus-like particles (Saenz et al., 2005). The TRIM5 proteins of humans and some Old and New World monkeys show species specificity in their ability to block infection of particular retroviruses following virus entry into the host cell (Song et al., 2005a). A potential risk with integrating gene therapy vectors such as the retroviral vector is the possibility of systemic propagation of replication-competent retroviruses, with the potential for disrupting expression of essential genes or the activation of otherwise silent promoter/enhancer regions (Ralph et al., 2006). Strategies that render the virus replication incompetent are in place, however, with recombinant lentiviruses.

V. SUMMARY

Gene therapy is an attractive approach for the treatment of a range of ocular diseases. It has several possible advantages

over the classical pharmacotherapies, such as providing a long-lasting therapeutic effect after a single treatment. This could circumvent the issue of patient compliance with multiple injection or topical drop therapies. Nevertheless, significant challenges, such as safe and efficient gene transfer into target cells/tissues, remain before gene therapy can be used to treat ocular disease in humans. Continued research to screen specific genes and proteins, enhance delivery vectors, minimize unfavorable immune responses, and establish more suitable animal models, will help realize the potential clinical applications of this promising new therapeutic strategy.

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