Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

immunopurifying the PLAP-expressing neurons, it was possible to analyze their mRNAs and identify additional genes that may be involved in dopaminergic amacrine cell function (Gustincich et al., 2004). A similar transgenic approach has recently been used to generate a mouse line expressing Cre recombinase in dopaminergic neurons in the retina and brain (Gelman et al., 2003).

Ever since the original descriptions of cholinergic amacrine cells (also called starburst amacrine cells), there has been debate as to the physiological function of these cells (Hayden et al., 1980; Masland et al., 1984). Starburst amacrine cells have radially symmetrical dendritic arbors that stratify in either of two narrow zones within the IPL, they release GABA as well as acetylcholine, and they have a retinal coverage factor estimated to be between 10 and 70, depending on the species. Because of the unusual distribution of inputs and outputs and their costratification with the two arbors of direction-selective (DS) RGCs, starburst amacrine cells have long been thought to be involved in DS responses to moving stimuli. A variety of approaches have been taken to test this hypothesis. In one approach, calcium imaging of DS responses in the dendritic arbors of starburst amacrine cells showed that these arbors have highly asymmetrical responses to stimuli moving across the retina in different directions (Euler et al., 2002). A second approach used a genetic tagging technique to ablate starburst amacrine cells from the mouse retina and then determined the effect of this ablation on DS RGC responses (Yoshida et al., 2001). A human interleukin-2 receptor α subunit (hIL- 2Rα)–GFP fusion protein was expressed as a transgene under the control of the mGluR2 promoter. One of the transgenic lines expressed hIL-2Rα–GFP only in starburst amacrine cells, a subset of the cells that express mGluR2. A monoclonal anti-hIL-2Rα antibody conjugated to a bacterial toxin was injected intraocularly in this transgenic line, resulting in an almost complete ablation of starburst amacrine cells. Concomitant with this ablation, there was an almost complete loss of DS RGC responses and an increase in the spiking rate of presumptive DS RGCs regardless of the direction of the stimulus. In addition, the optokinetic reflex was abolished, while pupillary constriction was preserved. This study demonstrated an essential role for starburst amacrine cells in the DS circuitry, and of DS RGC responses in the optokinetic reflex.

Conditional gene deletion has been used to define the role of cholinergic signaling by starburst amacrine cells in the spontaneous waves of activity that characterize the developing retina. Cre-mediated recombination can be induced throughout much of the retina by using a transgene expressing Cre under the control of an intronic enhancer from the Pax6 gene (Pax6αCre) (Marquardt et al., 2001). This transgene is active in proliferating retinal precursors beginning at embryonic day E9.5, prior to the differentiation of all retinal

cell types. Interestingly, the expression pattern of this transgene excludes a wedge-shaped domain in the center of the retina. Using the Pax6αCre line, it is possible to ablate any loxP-flanked gene in the retina while largely avoiding the deleterious effects of gene deletion in the rest of the embryo. To eliminate cholinergic signaling in the retina, the gene encoding choline acetyltransferase (ChAT), an enzyme required for the synthesis of acetylcholine, was deleted using the Pax6αCre line (Stacy et al., 2005). ChAT is expressed in all cholinergic neurons in the body, but in the retina it is expressed only by starburst amacrine cells. This elimination of retinal cholinergic signaling had little or no effect on retinal morphology but delayed by several days the appearance of the spontaneous waves of activity. It would be interesting to investigate the long-term effects of this manipulation on DS RGCs.

In the context of new genetic tools for visualizing neurons, special mention should be made of mouse lines carrying fluorescent proteins under the control of the Thy-1 promoter (Feng et al., 2000). Thy-1 is a GPI-anchored cell-surface protein of uncertain function that was originally identified on thymocytes (T cells) in both mice and humans. It was subsequently found in the retina, where it decorates the surface of all or nearly all RGCs. Feng and colleagues made the extremely useful observation that transgenic lines in which any of a variety of fluorescent reporters—GFP, CFP, YFP, RFP—are driven by a Thy-1 promoter typically show reporter expression in relatively small numbers of neurons, with different lines expressing the reporter in different neuronal subtypes. Each of these lines generally carries multiple transgene copies integrated at a single site, and, perhaps because of this high copy number, several of the lines produce readily detectable fluorescence in both large and small arbors in those retinal neurons that express the transgene. The sparse expression that is a characteristic of the Thy-1 transgenic lines is also important for tracing the full extent and ramification of individual arbors. In one application of this system, the GFP-M line has been used to visualize a variety of wide-field amacrine cells that would have been difficult to completely fill by tracer injection (Lin and Masland, 2006).

Retinal ganglion cells

RGCs are the projection neurons of the vertebrate retina. They have been a focus of intense interest for over a century not only because they encode and transmit all of the information captured by the eye but also because they offer an excellent system for studying axonal pathfinding and target selection by projection neurons (Hartline, 1938; Kuffler, 1953; Attardi and Sperry, 1963; Schnitzer and Meister, 2003). At the time of this writing, a literature search with the key words “retinal ganglion cell” and “mouse” yielded 178 papers, of which 140 were published in the past 10 years.

Although a majority of these studies focus on RGC development, the ability to discriminate individual RGCs using genetic techniques will likely lure an increasing number of physiologists and systems neuroscientists to ask their favorite questions in this system. Several classification systems for RGC cell types derived by genetic and nongenetic approaches have been published (Sun et al., 2002; Badea and Nathans, 2004; Kong et al., 2005; Coombs et al., 2006). Here we describe several genetic approaches that have proved useful in the study of RGCs.

It has been known for some time that ipsilateral RGC projections are diminished in albino mice (Guillery et al., 1973). To more precisely assess the role of melanin (or lack of melanin) in RGC axonal pathfinding, Cronin et al. (2001, 2003) adapted the components of the lac repressor-lac operator system, one of the best-studied prokaryotic gene regulatory systems, to generate an inducible gene expression system in the mouse. In E. coli, the lac repressor protein (lacI) binds to and inhibits transcription from promoter elements containing the lac operator. This transcriptional repression can be abolished by addition of isopropyl-thiogalactoside (IPTG), a synthetic lactose analogue that binds the repressor protein and decreases its affinity for the operator. Cronin and colleagues synthesized a DNA segment encoding the lacI gene (the natural E. coli sequence is extensively methylated and transcriptionally silenced when introduced in mammalian cells) and then created transgenic mice in which the synthetic lacI DNA (synlacI) is under the control of the chicken β-actin promoter, leading to its expression in many tissues and cell types. These transgenic mice were crossed to a line carrying a tyrosinase transgene with three lac operators inserted near the promoter. In the absence of the synlacI transgene, the tyrosinase transgene produces tyrosinase, and therefore induces coat and retinal pigmented epithelium melanin production (i.e., pigmentation) in an albino genetic background. In the presence of the synlacI transgene, coat color and eye pigmentation revert to the albino phenotype. However, if IPTG is administered in the food, the tyrosinase transgene is derepressed, resulting in pigmentation. Using this system, the authors were able to demonstrate that tyrosinase activity is required during an early time window when RGC axons are growing (Cronin et al., 2003).

The Thy-1-GFP-H line, similar to the M line mentioned earlier in the context of amacrine cell analysis, has also proved useful in the study of RGC morphology. In one study, RGC dendritic morphologies were quantified during the period surrounding eyelid opening, and the effect of dark rearing on the stratification of RGC dendritic arbors within the IPL was measured (Tian and Copenhagen, 2003). It was found that in P10 retinas (just before eye opening), about 50% of labeled RGCs were bistratified, and that under normal light exposure conditions this number decreased to 30% by P30. However, when animals were reared in the

dark, the fraction of labeled RGCs that were bistratified was still around 50% at P30, indicating that light exposure plays a role in RGC stratification or differentiation. These data were further supported by multielectrode array recordings which showed a decrease in the number of ON-OFF RGCs between P10 and P30 under normal rearing conditions and the persistence of ON-OFF RGCs in dark-reared animals.

By crossing the Thy-1-GFP alleles with other transgenes expressing fluorescent proteins, one can begin to ask questions about the wiring of various cell types, such as bipolar cells and RGCs (Lin and Masland, 2005). Moreover, crossing these transgenes into various gene knockout backgrounds can provide insights into the development of the labeled cell types (Lin et al., 2004). We note that one potential challenge with a strategy of this type is that the promoter that drives the transgenic reporter may be under the control of the gene that is knocked out, in which case a change in transgene expression may reflect this regulatory relationship rather than a change in the fate of the labeled cell.

An especially useful reporter for the study of projection neurons is the τ-β-galactosidase fusion protein (tau-lacZ). Tau is a microtubule-associated protein, and as a result, taulacZ nicely labels axonal microtubules. The tau-lacZ reporter has been knocked into the genes encoding several transcription factors expressed in RGCs (Pak et al., 2004; Pratt et al., 2004; Quina et al., 2005), as well as the gene encoding melanopsin, a photopigment expressed in a subset of RGCs (Hattar et al., 2002). In melanopsin tau-lacZ knock-in mice, β-galactosidase accumulation has been used to catalogue the central targets for these RGCs (Hattar et al., 2006).

Genetic cell-labeling techniques have also made contributions to our understanding of RGC physiology. For example, reporters of intracellular calcium have been expressed in RGCs using the tetracycline-inducible system (Hasan et al., 2004; described in the next section), allowing real-time measurements of intracellular calcium in conjunction with the electrical activity of the cells. These genetically encoded fluorescent reporters should facilitate optical measurements across large cell populations.

Genetic systems for site-specific recombination and drug-controlled gene expression

The Cre-lox recombinase system has been noted at several points in the preceding sections. An application of the Crelox technology that was not previously discussed involves using site-specific recombination as an indelible marker of lineage. In one version of this lineage analysis, the Cre coding region is inserted into a gene that is expressed in the progenitors of a particular lineage, for example a gene encoding one of the basic helix-loop-helix (bHLH) transcription factors that are transiently expressed during neural development. By crossing in a reporter that is expressed only

following Cre-mediated deletion of a loxP-flanked transcription termination signal, one can visualize all the progeny of the cells in which the bHLH gene was previously active (Zirlinger et al., 2002; Yang et al., 2003; Ma and Wang, 2006).

A somewhat more flexible version of this approach uses a Cre-recombinase that is fused to the ligand-binding domain of a mutated estrogen receptor (ER(T)) that recognizes the synthetic ligand 4-hydroxytamoxifen (4-HT) instead of the endogenous ligand estrogen (Feil et al., 1996; Brocard et al., 1997). The Cre-ER(T) fusion protein is sequestered in the cytosol in the absence of 4-HT; systemic or local administra-

tion of 4-HT releases the fusion protein, which then migrates to the nucleus and catalyzes site-specific recombination (figure 49.3B). By using CreER(T), the timing of the sitespecific recombination event can be controlled to within 1 day at any time in the life of the mouse simply by administering 4-HT; for prenatal exposure, 4-HT is administered to the mother. The efficiency of this process depends on several factors: (1) the abundance of the CreER(T) fusion protein,

(2) the availability of the target locus for recombination, which appears to be determined by local chromatin environment, (3) the amount of 4-HT delivered, and (4) the differentiation state of the target cell.

A

Cell specific promoter rtTA

Figure 49.3 Pharmacological control of gene expression. Pharmacological approaches can be used to express genetic elements with improved spatial and temporal control or in a more restricted cell population. A, A transgene driven by a cell-specific promoter expresses rtTA, a doxycycline-sensitive transcriptional regulator. Unliganded rtTA is inactive but can be activated by the administration of doxycycline, causing it to bind to a specific DNA recognition site (tetO), and induce the transcription of a second genetic element. Thus, by generating one cell-specific transgene and crossing it to various rtTA-sensitive reporters, one can activate a reporter or a functional modulator of the target cells in a timed fashion, depending on doxycycline administration. B, A target locus (controlled by promoter 2) can be modified by the activity of a drug-sensitive Cre

recombinase, CreER(T), expressed under the control of promoter 1. CreER(T) is produced in an inactive form and is activated by the administration of 4-HT (4-hydroxytamoxifen). The active recombinase then deletes open reading frame (ORF) 1, allowing ORF 2 to be transcribed under the control of promoter 2. This strategy can be used for conditional gene ablation, if the second locus is arranged as shown in figure 49.2, or for the conditional overexpression of reporter genes or other elements. Since the final pattern of ORF 2 expression is controlled by the spatial intersection of the expression patterns of promoters 1 and 2, as well as by 4-HT, this combinatorial strategy exhibits great flexibility for labeling and manipulating neuronal targets.

In an initial application of this method for lineage tracing, a CreER(T) line with ubiquitous expression was generated by knocking CreER(T) coding sequences into the ROSA26 locus (Zambrowicz et al., 1997; Badea et al., 2003). ROSA26 is a genetic locus identified by Soriano and colleagues during an insertional mutagenesis screen. The ROSA26 locus becomes transcriptionally active early in development, and transcription persists in all or nearly all cells in the body from that time on. Crossing the ROSA26-CreER(T) line to one that carries a ubiquitously expressed and Cre-activated PLAP (the Z/AP line constructed by Lobe et al., 1999) permits histochemical identification of those cells that have inherited a Cre-mediated recombination event (Badea et al., 2003). As expected, administering 4-HT at earlier or later developmental times generates, respectively, larger or smaller clones. Also as expected, the ROSA26-CreER(T);Z/AP combination generates labeled clones throughout the body at a density that depends on the 4-HT dose. Lineage tracing with ROSA26-CreER(T);Z/AP revealed the previously defined patterns of radial clones of retinal neurons (Price et al., 1987; Turner et al., 1990), and it also showed that retinal capillaries are composed of the intermingled progeny of a relatively small number of endothelial progenitors (Badea et al., 2003).

A second application of the CreER(T) technology is in the production of tissues with very low densities of genetically labeled neurons for morphological analysis. This approach has been used to generate a catalogue of all the major neuronal cell types in the retina by using ROSA26-CreER(T);Z/ AP mice with both systemic and intraocular 4-HT injections (Badea and Nathans, 2004). The resulting set of labeled neurons has provided the raw material for a quantitative analysis of the patterns of stratification and dendritic arborization for all the major cell types. For example, in analyzing the arbors of polyaxonal amacrine cells, the data show that the lengths of individual dendrites can be comparable to the diameter of the retina, a finding also obtained with Thy-1 transgenic mice (Lin and Masland, 2006). The ROSA26-CreER(T);Z/AP labeling method has more recently been applied to the study of neuronal morphologies in various mutant retinas (Badea and Nathans, unpublished results).

Finally, we note that reversible drug control of gene expression, as described for synlacI/IPTG control of melanin production, is still a largely untapped technology for visual system studies. The rtTA is currently the system most widely used in both mammalian cell cultures and mice (Urlinger et al., 2000; Hasan et al., 2004; figure 49.3A). In this system, the tetracycline repressor (TetR), which in E. coli maintains the gene encoding the tetracycline efflux pump in a repressed state in the absence of tetracycline, has been engineered to bind rather than release its DNA target in the presence of tetracycline or related drugs, such as doxycycline (hence the

adjective “reverse” in rtTA). The rtTA protein also carries a eukaryotic transcriptional activation domain, and therefore, when it binds to its DNA target (tetO), previously engineered into the gene of interest, transcription ensues. Coding sequences for rtTA, as well as for Cre, have recently been introduced into transgenic mice under the control of the Thy-1 promoter (Campsall et al., 2002; Kerrison et al., 2005).

Conclusion

We hope that the examples presented here make a convincing argument for using the mouse as a genetic model organism for the study of visual system function and development. However, the examples also illustrate the need to develop additional tools to target more refined subpopulations of neurons. We anticipate that combinations of gene expression cassettes with partially overlapping expression patterns—for example, one cell type–specific promoter controlling CreER(T) and a second cell type–specific promoter controlling a Cre-dependent reporter—will be developed in the near future to provide improved specificity by the intersection of their expression patterns. These and other advances should ultimately provide investigators with the ability to precisely control both cell type and temporal patterns of neuronal gene expression.

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A variety of therapeutic strategies aimed at combating inherited retinal diseases have recently been developed. In general, these strategies can be divided into three categories: gene therapy, pharmacological neuroprotection, and stem or precursor cell therapy. Of these approaches, only gene therapy is capable of curing a disease state. The others seek either to halt or slow the progression of an existing disorder or to replace key retinal cells once they are lost or become dysfunctional. Of course, if tissue replacement utilizes a sufficient number of normal cells, a cure may also be effected. This chapter focuses on gene-based therapeutic approaches for treating retinal degeneration (RD) in mouse models.

Gene therapy vectors

The history of gene therapy is best illustrated by the attempts to develop a lasting treatment for cystic fibrosis (CF). The earliest CF gene therapy trials utilized recombinant adenoassociated virus (AAV), which was found to be ineffective, owing to problems with delivery to the target cell and insufficient expression levels (Griesenbach et al., 2006; Flotte et al., 2007). In addition to AAV, two other candidate gene therapy vectors, adenovirus and lentivirus, were investigated for their therapeutic potential for treating CF. However, the occurrence of two high-profile adverse events in humans involving these vectors affected their utility in CF and other clinical studies. The systemic administration of an adenovirus vector for ornithine transcarbamylase (OCT) deficiency in which adenovirus vector was delivered to the liver resulted in a fatality (Raper et al., 2003). It was concluded that the high dose of vector (required because of poor expression from vector) given to this patient was immunogenic. Additionally, administration of a lentiviral vector to patients with X-linked severe combined immunodeficiency syndrome resulted in site-specific integration of the vector near a specific pro-oncogene, which resulted in three cases of leukemia, one of which was ultimately fatal (Williams, 2006). AAV’s relatively nonimmunogenic properties and apparent

lack of chromosomal integration indicated that it was inherently safer and more effective than the aforementioned viral vectors. Newer AAV vectors have been designed to address earlier shortcomings and are now in the process of being evaluated for the treatment of CF (Flotte et al., 2005). These same advances in AAV vector technology are being applied to the gene therapy of various other disorders, including disorders of the eye (Warrington and Herzog, 2006).

Adenoassociated virus vectors

Although there are a number of strategies for delivering genes to cells, the nature of the target tissue and the therapeutic requirements for the disease normally dictate which choice is optimal. Viruses of the Parvoviridae family have shown the most promise in gene therapy, particularly for retinal disease. A member of this family, human AAV has been widely exploited as a vector for gene delivery because of its advantages over other viral gene therapy vectors, namely, safety, long-term expression, the ability to transduce terminally differentiated cells, and selective (as well as broad) tropism through the use of the numerous AAV serotypes currently available. AAV is a nonpathogenic, replicationdeficient dependovirus that contains a single-stranded genome of 4.7 kb flanked by inverted terminal repeats (ITRs), each 145 bases in length (Srivastava et al., 1983; Flotte and Berns, 2005). Specific sites within these ITRs control the conversion of single-stranded AAV genomes to their double-stranded DNA state necessary for subsequent transcription and translation (Qing et al., 1997, 1998; Mah et al., 1998).

Wild-type AAV is not associated with any pathological condition in humans. Recombinant AAV (rAAV) is produced by removing all native AAV coding sequences, leaving only the short ITRs flanking the promoter and cDNA of interest. This eliminates the virus’s ability to integrate site specifically into the genome; the integration events that do occur are very infrequent and require chromosomal breakage (Miller et al., 2004). Thus, the overall risk of an AAV

integration event activating an oncogene is considered low. AAV vectors do, however, promote long-term transgene expression by remaining in the transduced cell in a circular, double-stranded episomal form (Song et al., 2001, 2004; Duan et al., 2003). In other words, AAV-delivered DNA remains in the host cell nucleus as an independent genetic unit and not as part of the host cell’s genome. In vivo expression has been documented to persist for more than 6 years in a dog model of Leber congenital amaurosis (Acland et al., 2005; G. Acland and G. Aguirre, pers. comm., 2007). Additionally, AAV vectors have been shown to efficiently transduce terminally differentiated, nondividing cells. This is clearly vital for treating retinal diseases, most of which involve malfunction of photoreceptors or other terminally differentiated retinal cells.

There are more than 100 different variants of AAV, categorized into serotypes and genomovars (Gao et al., 2004). Among these genomic variants is a broad diversity of AAV serotypes that utilize a range of different receptors (Flotte and Berns, 2005). The nine AAV serotypes currently in wide use, AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, and 9, differ from each other to varying degrees in their capsid protein sequence critical for serotype determination. Capsid variations confer distinct tissue and cell affinities and, as a result, define the speed of expression onset and the overall intensity of transgene expression by AAV vectors. AAV serotype 2 (AAV2) was the first used for gene transfer to the rodent retina via subretinal injection and was subsequently shown to promote broad transduction of photoreceptors and retinal pigmented epithelium (RPE) (Bennett et al., 1997; Flannery et al., 1997; Ali et al., 1998). Subsequently other serotypes have shown potential for targeting transgene expression to specific subsets of cells in the mouse retina. Typically, non-serotype 2 AAV vectors are made by packaging the vector DNA flanked by serotype 2 ITRs into the desired capsid serotype, a process termed pseudotyping. Almost all non-serotype 2 vectors to date have been made by this technique. AAV5, like AAV2, targets photoreceptors and RPE after subretinal injection, but does so with greater efficiency than AAV2 (Yang et al., 2002). AAV1 and AAV6, two closely related serotypes, have both been shown to transduce primarily RPE (Xiao et al., 1999). AAV4 is the only serotype to be expressed solely in RPE, based on results in rat, dog, and nonhuman primate, and would therefore be expected to do the same in mouse (Weber et al., 2003). AAV3 does not appear to transduce retinal cells at all after subretinal injection (Yang et al., 2002). Although AAV7 and AAV8 have not been as comprehensively evaluated in the mouse retina as other serotypes, there is some evidence that both transduce RPE and photoreceptors, with AAV8 being more efficient (Lauramore, 2004). The transduction characteristics of AAV9 in mouse retina have yet to be reported in the literature. The use of ITRs from serotypes other than AAV2 has been

reported for an AAV5 capsid containing serotype 5 ITRs (Yang et al., 2002). This vector had a similar transduction pattern to that for AAV5 pseudotyped with serotype 2 ITRs, in that it was more efficient at targeting photoreceptors and RPE than standard AAV2.

When injected into the vitreous of mouse and rat, AAV2 has proven to be the most efficient serotype for targeting expression to the inner retina, primarily retinal ganglion cells (RGCs; Ali et al., 1998; Auricchio et al., 2001; Liang et al., 2001a; Martin et al., 2003). In addition to targeting expression to the inner retina, limited spread of expressed transgene to the optic nerve and brain has also been observed (Dudus et al., 1999). The transduction patterns of various AAV serotypes following subretinal or intravitreal delivery are summarized in table 50.1.

In addition to serotype selection, promoter choice aids significantly in defining the retinal cell specificity of transgene expression. For broad transgene expression in the retina, the cytomegalovirus (CMV) immediate early promoter has historically been used (Ali et al., 1998). More recently, the chimeric CMV-chicken β-actin promoter/ CMV enhancer (CBA) has been used in applications where high-level, long-term expression of protein in a broad variety of cells is required (Raisler et al., 2002; Pang et al., 2006). In cases where a single cell type is the target of therapy, nonviral cell-specific promoters enhance the safety of AAV vectors by reducing possible toxicity associated with the transduction of nontarget cell types. AAV-mediated expression targeted to photoreceptors was first achieved in rat using 472 bps (−386/+85) of the proximal mouse rhodopsin promoter (Rho, also commonly referred to as mOP)

Table 50.1

Transduction characteristics of various AAV serotypes following subretinal or intravitreal administration to the mouse retina

* The first number indicates the serotype of the ITRs flanking vector DNA, and the second number represents the serotype of capsid protein.

in conjunction with AAV2 (Flannery et al., 1997). It has subsequently been shown to be the case for mouse as well (Min et al., 2005; Pawlyk et al., 2005). Some controversy exists as to whether mOP targets both rods and cones, but several recent studies have provided convincing evidence that cones are indeed transduced when using this promoter, particularly in conjunction with AAV5 (Glushakova et al., 2006a; Haire et al., 2006). A human blue cone opsin promoter originally shown to preferentially target cone photoreceptors in rat has also been shown to effectively target cones in mouse retina when used with AAV5 (Glushakova et al., 2006b). In addition, a human red cone opsin promoter fragment used in conjunction with AAV5 has also been shown to preferentially target cones in mouse retina (Alexander et al., 2005). Promoter analysis indicates that RPE-specific expression can be achieved using portions of the vitelliform macular dystrophy 2 (VMD2) promoter of the bestrophin gene (the gene responsible for Best disease) or the RPE65 promoter (Boulanger et al., 2000, 2002; Esumi et al., 2004, 2007). This has been confirmed in mouse when these promoters have been used in conjunction with AAV1 (Glushakova and Hauswirth, 2004).

Regulatable systems of gene expression are desirable in cases where continuous, high levels of expression of a therapeutic agent may be harmful to retina. Two have been used in conjunction with AAV vectors targeted in the retina: the tetracycline-inducible and the rapamycin-inducible transcriptional regulatory systems. Although both have been studied in rat (McGee-Sanftner et al., 2001; Auricchio, Rivera, et al., 2002; Smith et al., 2005), it is reasonable to expect that the results are equally applicable to mouse.

This chapter focuses on the application of AAV-mediated gene therapy in several different mouse models of inherited RD. In all studies mentioned, AAV-mediated somatic gene transfer resulted in significant functional improvement, as assessed by electroretinography (ERG) or behavior, or by regeneration or stabilization of retinal structure. In some cases, knowledge gained from initial work in mice has set the stage for the development of human clinical trials. A comprehensive list of these studies and the type of vector, promoter, and transgene used is given in table 50.2.

Antiangiogenic gene therapy

Although many clinical RDs are the result of mutations in genes expressed in the retina, some are secondary to systemic disease. The mammalian retina is one of the most metabolically active tissues in the body and therefore demands very high levels of oxygen via both the retinal and choroidal blood vessels. Systemic disease such as diabetes or retinopathy of prematurity (ROP) can result in retinal hypoxia that subsequently leads to retinal neovascularization. Age-related macular degeneration (AMD), the leading

cause of blindness in developed countries, in its “wet form” is characterized by neovascularization originating in the choroidal vasculature. Normal retinal and choroidal vessel beds are maintained by a balance of several endogenous proteins, including positive growth factors such as vascular endothelial growth factor (VEGF) and antiangiogenic proteins such as pigmented epithelium–derived factor (SERPINF1, more commonly referred to as PEDF). Traditional treatments (laser, photodynamic therapy) seek to delay the progression of the disease by destroying new, pathogenic blood vessels. They do not, however, address the underlying cause, that being the recurring, inappropriate proliferation of the retinal or choroidal vasculature. The delivery of proteins with antiangiogenic properties has proved somewhat successful in patients with various neovascular or neovascu- lar-associated diseases (O’Reilly et al., 1994, 1997; Stellmach et al., 2001). Two such therapies currently available to patients with neovascular AMD are Pfizer’s Macugen (pegaptanib), a pegylated aptamer of VEGF, and Genentech’s Lucentis (ranibizumab), an anti-VEGF antibody. However, both require repeated intraocular injections for extended periods of time, thus posing risk and compliance issues for patients. In animal models, AAV-mediated gene transfer of antiangiogenic proteins to various cells of the retina has been employed to combat both retinal and choroidal neovascularization, thus suggesting AAV-vectored genes may be an attractive, long-term alternative for the treatment of local neovascular disease.

Therapy for retinal neovascularization

The oxygen-induced retinopathy (OIR) mouse model, more commonly referred to as the ROP mouse, has been developed for the study of retinal neovascular disease (Smith et al., 1994). Newborn mice are placed in a hyperoxic (ca. 75% oxygen) chamber for 5 days. This high-oxygen environment induces retinal capillary ablation, which, when mice are returned to normoxic conditions, results in relative hypoxia and retinal ischemia. This in turn leads to VEGF-mediated retinal neovascularization like that seen in humans with neovascular retinal disease.

A variety of antiangiogenic treatments have been tested in the ROP mouse. In one study, AAV2 was used to deliver PEDF, under the control of the CBA promoter, to the intravitreal or subretinal space of a newborn mouse eye (Raisler et al., 2002). The contralateral control eye received an equivalent injection of PBS. Retinal neovascularization was quantified in both treated and untreated (contralateral) eyes by counting the number of vascular endothelial cell nuclei above the inner limiting membrane in P17 eyes. The number of neovascular nuclei observed in eyes treated with AAV2- PEDF was significantly reduced relative to that in control eyes. Similar results were obtained when AAV serotype 1