Ординатура / Офтальмология / Учебные материалы / Retinal Vascular Disease Joussen Springer

180 I Pathogenesis of Retinal Vascular Disease

10 I

Ultrasound has been shown to increase transient permeability of cell membranes. By using contrasting agents (Optison), delivery of the plasmid DNA can be greatly enhanced compared to without. Ultrasound is a commonly used imaging aid in ophthalmology, so its use may have practical applications.

Magnetofection relies on magnetic nanoparticles that surround the DNA. These complexes are then guided by magnetic fields to place them in close proximity to the targeted cells. There has been preliminary success with this application both in vitro and in vivo with DNA uptake several hundred times higher than naked DNA alone. This technique is fairly novel and long-term risks associated with using these nanoparticles have yet to be determined.

Bioballistic delivery involves coating the DNA with gold particles, which can then be delivered by a “gene-gun.” The DNA is basically shot into the desired tissue via high-pressure air. This method has been used for DNA vaccination but normally results in very low transient expression of the transgene.

10.1.3 Target Diseases

Essentials

A small number of retinal vascular diseases account for most blindness in developed countries (Fig. 10.1.6.)

Molecular advances are continually elucidating the pertinent biologic pathways and identifying the genes involved, thus offering possible targets for gene therapy (see Table 10.1.2)

10.1.3.1 Retinopathy of Prematurity

Retinopathy of prematurity (ROP) accounts for a significant proportion of childhood blindness in developed countries. The disease is classified as having two independent stages [25].

Stage 1: Normal vascularization occurs in utero at month 4 of gestation. Premature birth and standard oxygen therapy halts ocular vasculogenesis, leaving a considerable portion of the eyecup avascular.

Stage 2: Return to environmental normoxia stimulates a hypoxic/ischemic response in the eye. The avascular zone of the retina becomes metabolically active, initiating several factors including VEGF to be upregulated and inducing a neovascular state. This in turn results in aberrant blood vessel formation and pathology similar to that seen in proliferative vitreoretinopathy.

Current treatments involve either cryoor laser ablation (the latter is standard) of the avascular zones in order to prevent further neovascularization. Treatment success is dependent on the severity of retinopathy and the number of zones involved [11].

10.1.3.2 Age Related Macular Degeneration

Age related macular degeneration (AMD) is the leading cause of blindness in patients over the age of 60 in the developed world [29]. Its exact etiology is not fully understood and it is considered to be a multifactorial disease, associated with both environmental and genetic stimuli. Patients with vascular AMD present with drusen, geographic atrophy, subretinal neovascularization, and central vision loss [16]. Extensive clinical trials have led to the development of preventive measures such as dietary supplements [24] and life-style risk assessments [12]. Despite this, AMD continues to be a major cause of morbidity in the elderly, a steadily growing population. Currently, treatment options include surgery, photocoagulation, and photodynamic therapy and the injection of a variety of anti-angiogenic substances [5].

10.1.3.3 Proliferative Diabetic Retinopathy

Proliferative diabetic retinopathy (PDR) is associated with uncontrolled hyperglycemia as a result of diabetes mellitus and is a leading cause of blinding

disease in patients under age 30. The proliferative process is intimately related to the vascular structure of the retina, specifically the interactions between the endothelial cells and the pericytes [9]. Pathology is believed to involve the breakdown of the bloodretinal barrier, which then leads to vascular permeability, macular edema and neovascularization. The exact cause of this breakdown is not entirely understood but may involve endothelial tight gap junction collapse with subsequent introduction of blood sugar by-products (advanced glycosylation end products) that stimulate pericyte death and the upregulation of growth factors [26].

10.1.3.4 Retinal Vaso-occlusive Disorders

A leading cause of blindness in patients over age 60 is retinal vaso-occlusive disease. Occlusions can affect either the veins or arteries that supply the outer retina. Central retinal artery occlusions (CRAO) and central retinal vein occlusions (CRVO) often create the most significant disease states compared to peripheral branch obstructions.

CRAO: Results in abrupt and massive visual loss, with visual outcomes of 20/200 or lower [30]. Occlusion of the central retinal artery or branch arteries can have a variety of etiologies ranging from transient nighttime hypotension to cardiovascular disease and

diabetes. CRAO appears to be more com- 10 I mon in males than females and is usually

diagnosed too late for effective therapeutic intervention. The time required for irreversible loss of vision in an experimental model of CRAO was less than 250 min [10]. The ensuing pathology is believed to be associated with ischemic initiation of the apoptotic cascade [30] with loss of retinal function, namely a significant reduction in ganglion cell layer response during the electroretinogram (ERG) [15]. The ischemia also results in neovascular complications.

CRVO Considered the most prevalent vaso-occlu- or sive disorder in the US, it results from BRVO: blockage in the central retinal vein or

branch veins. It is classified as having two distinct forms (ischemic and non-ischemic) that initiate cell death much more slowly than CRAO. Non-ischemic CRVO is normally of acute onset and has a far better visual outcome than the ischemic form. Ischemic CRVO is associated with retinal hemorrhage, tortuous veins, cotton wool spots, and markedly decreased visual acuity with poor prognosis. The underlying derivation of CRVO is multifactorial (high cholesterol, emboli, and inflammatory disease may all play a role) with patients over age 50 most susceptible. Current treatments for CRVO involve anticoagulants and steroids.

10.1.4. Fundamentals of Gene Therapy

Essentials

In vitro experimentation is paramount to gene therapy advancement

Angiogenic models can emulate in vivo conditions

There are two approaches for gene delivery to the body

Cell derivation...does this matter?

10.1.4.1 In Vitro

In vitro technologies are the foundation of basic science research and have led to the most detailed understanding of retinal pathology. In vitro experiments usually involve specific cell cultures or tissues masses. Cells germane to the study of retinal vascular disease have for the most part been established and

are commercially available. Several endothelial cell properties such as cell migration, tube formation, and proliferation can be assayed to study angiogenesis. There are a number of beneficial tissue-based assays that have been developed to bridge the gap between cellular and animal research, such as the aortic ring and chick embryo assay. The advantage to these types of experiments is the ability to control one has over many key parameters. For example, environmental parameters such as hypoxia are possible via hypoxia chambers and incubators offering the ability to mimic pathological conditions using premixed gas preparations.

At one time, vectors for experimental use had to be engineered and prepared using laborious methods. Today, commercially available kits including detailed protocols and reagents make vector production practical and affordable for most laboratories. Kits are currently available for producing adenoviral, lentiviral, and AAV-based vectors.

10.1.4.2 Ex Vivo Versus In Vivo

There are two approaches to introduce therapeutic genes into the body. The ex vivo approach entails removing a cell or tissue type, delivering a gene, and reintroducing those cells to the affected site. This approach allows cells to be transduced in culture and then transplanted to a target site, where they can mimic normal tissue and deliver therapy to the affected area. This was shown to be possible in the eye when retroviral-transduced RPE cells were transplanted under the retina and shown to inhibit VEGF-induced choroidal neovascularization in a rabbit model. This approach offers several potential advantages. The gene transfer can be monitored and tested for function, insertional events (dependent on delivery approach), and expression level. This provides the assurance that what will be introduced into the patient is what was initially desired. There are cases, however, in which this approach may not be feasible as diagnostically testing every adverse possibility is time-consuming and extreme situations may not afford this option. In such cases, in vivo delivery is the only option. Direct or local administration is considered far safer then systemic delivery and offers fewer possible complications. The advent of better targeting vectors will also improve in vivo administration in the future. This could be accomplished with the use of advanced viral pseudo-typing methods that would allow specific cells to be targeted based on outer membrane receptor profiles. Another solution relies on tight control of gene insertion. Placing a gene precisely where it is desired in the genome could afford greater safety and stability.

10.1.4.3 Somatic Cells Versus Stem Cells

Somatic cells are differentiated cells that comprise the majority of tissues in the body. To date, most gene therapy research has focused on gene transfer into somatic cells. These cells offer the advantage of being easy to acquire, biologically static, and potentially easier to control.

Gene therapy using somatic cells:

Can act as factories to produce a secretable form of the transgene-product, supplying surrounding cells with said “therapeutic” protein.

Can be targeted themselves, resulting in single cell effects such as with the use of suicide genes in cancer research.

Can be targeted to initiate cellular cascade events that stimulate surrounding cells via a bystander effect or activation of downstream products.

Stem cells are undifferentiated cells that become specialized given specific stimuli. Stem cells fall into three main categories, totipotent, pluripotent or multipotent.

Totipotent cells have the ability differentiate into any cell type and form complete organisms or tissues/organs.

Pluripotent cells are true stem cells but lack the ability to form extraembryonic membranes (i.e., placenta cells). Embryonic stem cells and germ line cells fall into this class.

Multipotent cells are true stem cells but can only differentiate into a subclass of specialized cells, for example hematopoietic stem cells that give rise to blood cells.

Totipotent and pluripotent gene transfer still remains a novel therapeutic strategy but offers the possibility of organ regeneration and gene replacement therapy during embryonic development. The use of multipotent cells, on the other hand, has much promise for use in retinal vascular gene therapy protocols. Multipotent cells possess specific cellular markers (i.e., CD34); therefore, it is possible to isolate the stem cells based on these characteristics with established methods. The isolated cells, such as hematopoietic stem cells (HSCs), can be transduced so as to harbor therapeutic genes and can be subsequently delivered to the retina by intravitreal injection. Recently this method resulted in direct targeting of endothelial progenitor cells (EPCs), a subset of HSCs, to retinal astrocytes. A marked inhibition of angiogenesis was observed after employing this technique in lineage negative EPCs derived from bone marrow using the T2-tryptophanyl-tRNA synthetase gene. The direct targeting and incorporation of these cells into newly developed blood vessels car-

rying therapeutic agents make this an attractive therapeutic approach. I 10

HSCs have been isolated from peripheral blood and bone marrow with comparable cell quality. The main differences between the two sources are cell yield and the procedures employed. Bone marrow contains a significantly higher proportion of CD34+ cells compared to peripheral blood. The use of cytokines has been shown to increase peripheral blood levels to that of bone marrow but long term risks associated with cytokine use are unknown. Bone marrow isolation is also an invasive procedure requiring anesthesia, wound recovery, and pain management. For these reasons peripheral blood may offer a more convenient means of harvest. Regardless of source, ex vivo gene transfer into hematopoietic stem cells is an excellent therapeutic prospect without the pitfalls associated with allograft transplantation.

10.1.5 Genes as Drugs/Clinical Trials

Essentials

Human gene therapy for ocular disease

Anti-proliferative genes enter the limelight

Human ocular gene therapy trials using adenoviral mediated gene transfer have shown efficacy for the treatment of retinoblastoma and ocular melanoma. Both treatments have employed the herpes simplex thymidine kinase gene for its cell suicide properties. Although these are not retinal vascular diseases per se, they do demonstrate the use of gene therapy in the eye. Recently, the first clinical trial use of an antiangiogenic gene for the treatment of a proliferative ocular disease has been demonstrated.

In 2004 – 2005, the use of adenoviral-mediated delivery of the PEDF gene for the treatment of neovascular AMD was reviewed in a Phase I clinical trial. This multicenter study sponsored by GenVec was performed to evaluate dose response in 28 patients diagnosed with neovascular AMD. The dose escalation study assessed viral load ranging from 106 to 109.5 viral particles. Side effects appeared to be minor, including mild inflammation and moderate transient intraocular pressure spikes. Patients were clinically evaluated 13 times over a 12-month period. Tests performed included a full ophthalmic exam, physical exam, blood chemistry, viral cultures, and viral protein ELISA. None of the treated patients showed evidence of viral replication from urine or sputum samples. There was also no consistent or robust pattern of anti-adenoviral reactive antibodies detected. Other observations suggested a dose

dependent reduction in lesion size over the study 10 I period. These observations, while not a specific trial endpoint, suggest two points: that a single treatment may provide sustained therapeutic results for several months and that the use of PEDF may benefit patients with neovascular AMD. Phase II clinical

assessment for this drug is underway.

10.1.6 Ethical/Safety Concerns

The past 20 years of scientific advances have led to the development of human gene therapy research. This same research has given rise to many bioethical and safety questions concerning the use of gene therapy and future implications. Gene therapy has both somatic and germ line implications. Currently, somatic based research abounds and although safety is an issue, it is less of an ethical quandary. On the other hand, germ line manipulation using gene transfer is a subject that raises many ethical questions.

What is normal variation? What is health?

Can genetic prevention lead to genetic “enhancement” or “selection“?

Should embryonic stem cells be used in research? In treatment?

To date, the use of germ line stem cells for ocular gene therapy has not been explored significantly; this discussion will focus on safety issues concerning gene transfer into somatic and multipotent stem cells.

Integrative vectors such as retroviruses pose the risk of transgene insertion into areas of the genome that would disrupt native gene expression. This can be detrimental in cases where downstream oncogenes are upregulated or when tumor suppressor genes can no longer function adequately. This fear was realized after a human trial for the treatment of X linked SCID resulted in three of the patients developing leukemia after T cell transduction.

Another potential adverse effect of gene therapy is related to immune response. The two main issues involve immune attack on vector components such as viral proteins or on the exogenous “therapeutic” gene product itself. First, viruses can encourage a significant immune mobilization; this was made tragically apparent after the death of a patient who was systemically administered a lethal dose of adenovirus. Since then, strict guidelines have been developed to prevent this type of complication. Second, gene replacement therapy in which a functional copy of a gene is introduced into the body could elicit immune responses because this form of the protein is immunologically novel to the body.

Ocular gene therapy may have an advantage over gene therapy at other sites in the body, as the eye is a compartmentalized structure that is essentially immunoprivileged. Experiments using ocular gene therapy do not generally give rise to immune complications, as discussed above in the description of the AdPEDF.11 clinical trial. For these reasons, the eye may be the most suitable organ in which to test and evaluate gene therapy. Advances in vector construction have and will continue to evolve addressing integrative issues and concerns.

References

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Core Messages

Norrie disease (ND) is an X-linked retinal dysplasia

The ND (NDP) gene encodes for norrin, a protein causing Norrie disease

Norrin is critically required for the formation of the inner retinal plexus and is involved in the

completion of the final secondary capillary layer, but does not include formation of the initial angiogenic sprouting

Norrie disease mutant mice demonstrate structural abnormalities in the retinal vasculature

10.2.1 Introduction

Norrin or Norrie disease protein is a 131-amino- acid-long, secreted protein that forms disulfidebonded oligomers, which associate with the extracellular matrix upon secretion [11]. Sequence analysis indicates a cysteine knot motif similar to that seen in growth factors of the cysteine knot family [8]. Norrin is predominantly expressed in brain, retina and the olfactory bulb [1, 3]. Mutations in the ND gene (NDP) that encodes norrin cause Norrie disease, an X-linked retinal dysplasia [2, 7]. The biochemical function(s) of norrin have been unclear for several years. Recent studies strongly indicate that norrin is critically required to induce the formation of the deep retinal capillary layers and the capillaries of the inner ear [10, 12, 19], a function that is mediated by high affinity binding to frizzled-4 (Fz4) and activation of the classical Wnt pathway [19]. In contrast to the function of other growth factors that modulate angiogenesis in the developing retina, norrin does not induce initial angiogenic sprouting, but is rather involved in the completion of a final secondary capillary layer.

10.2.2 Norrie Disease

Norrie disease (ND) is an X-linked, rare inherited disorder that presents with congential or early childhood blindness because of severe retinal dysplasia [16, 17, 18]. About one-third of individuals with Norrie disease develop progressive hearing loss and some degree of mental retardation. The classical

finding has been reported as a grayish-yellow, glistening, elevated mass that replaces the retina and is visible through a clear lens. These masses have been referred to as pseudoglioma. Vascular changes appear to be associated, including vascularization of the mass from the vitreous, often resulting in vitreous hemorrhage. The affected gene (NDP) is localized on chromosome Xp 11.3 and encodes for Norrie disease protein or norrin [2, 7]. Various mutations in NDP have been described (nucleotide deletions, nonsense, frameshift, splice site and missense mutations), which commonly appear to result in loss of function of norrin [1].

10.2.3 Norrie Disease Mutant Mice

To understand the molecular pathogenesis of Norrie disease, mutant mice with a targeted disruption of Ndp (Ndpy/–) were generated [3]. Ndpy/– mutant mice show fibrous masses in the vitreous body and an overall disorganization of the retinal ganglion cell layer [3]. The outer plexiform layer may disappear resulting in a juxtaposed inner and outer nuclear layer. In regions of juxtaposition, the outer segments of photoreceptors are no longer present. Starting from postnatal day 9, the number of retinal neurons in the inner retinal layers of Ndpy/– mutant mice substantially decreases as compared to wild-type littermates [13]. There are also pronounced structural abnormalities in the retinal vasculature, which include the complete absence of the two intraretinal capillary beds and the formation of vascular membranes at the vitreal surface of the retina [6, 12, 13]. In addition,

the hyaloid vasculature persists [6, 9, 13]. The changes in retinal structure correlate with a loss of function as dark-adapted ERGs in Ndpy/– mice show a dramatic loss of the positive b-wave, which is mostly shaped by the neurons of the inner retina, particularly the bipolar cells [10, 14]. In addition, no substantial oscillatory potentials, a set of higher frequency wavelets usually superimposed on the b- wave, are observed. Overall, ERG waveforms in Ndpy/– mice closely match those obtained in normal eyes in a state of retinal hypoxia [15]. An abnormal

vasculature has also been observed in the inner ear

of Ndpy/– mice, a finding that is consistent with the I 10 observation that the mice develop progressive hear-

ing loss up to profound deafness [12].

10.2.4 In Vivo Overexpression of Norrin

To learn more directly about the function of norrin, a mouse model with transgenic ocular overexpression of ectopic norrin by means of a strong lens-spe- cific promoter has been developed [10]. The pheno-

type of these animals indicates that norrin induces 10 I growth of ocular capillaries, as lenses of transgenic mice with ectopic expression of norrin show significantly more capillaries in the hyaloid vasculature that surrounds the lens during development. In vitro, lenses of transgenic mice in coculture with microvascular endothelial cells induce proliferation of the cells. These effects may be mediated through the additional action of other factors, as mRNA for vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) is found in higher amounts in the eyes of transgenic animals than in wild-type littermates. To learn if the ectopic expression of norrin could restore normal retinal vascular development and remodeling in Ndpy/– mice, the animals were crossbred with norrin overexpressing mice. In the resulting animals, both retinal capillary beds are present and can be visualized by conventional light microscopy, electron microscopy and labeling of vascular endothelial cells with biotinylated Griffonia (Bandeiraea) simplicifolia lectin I (Fig. 10.2.1). The capillaries show the typical ultrastructural characteristics of retinal capillaries, as they are covered by pericytes and surrounded by a complete basal lamina. In summary, the ectopic transgenic expression of lens-derived norrin completely restores normal vascular development of the retina in Ndpy/– mice. In addition, no abnormal vascular sprouting or signs of retinal neovascularization are observed, and the formation of abnormal dense vascular membranes at the inner retinal surface of

Ndpy/– mice is completely prevented.

The improvement in structure correlates with restoration of neuronal function in the retina, as both the positive b-wave and the oscillatory potentials are completely restored.

It is of interest to note that ectopic norrin not only restores normal capillarization of the retina, but also prevents the progressive loss of retinal ganglion cells (RGCs) seen in Ndpy/– mutant mice. A possible explanation is that hypoxia induces RGC death in Ndpy/– mice, and that the restoration of retinal capillaries caused by the presence of ectopic norrin maintains normal oxygenation of the retina. Still, a more direct neurotrophic effect of norrin appears to be possible, as transgenic mice with ectopic expression of norrin show more BrdU-labeled retinal progenitor cells at embryonic day 14.5 and thicker retinas in postnatal life.

10.2.5 Conclusion

Ectopic norrin induces growth of retinal capillaries, an effect that completely respects the normal vascular architecture of the retina and does not involve abnormal vascular sprouting or signs of retinal neo-

vascularization. An intriguing aspect of this observation is that pharmacologic modulation of norrin might be used not only for treatment of the vascular abnormalities associated with Norrie disease, but also for other vascular disorders of the retina [5, 10]. The expression of norrin’s receptor, Fz4, has been found in cultured human dermal microvascular endothelial cells and in endothelial cells from adult lung capillaries isolated ex vivo [4], indicating that ectopic norrin and Fz4 signaling could also be used for treatment of vascular disorders outside the eye.

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10.Ohlmann A, Scholz M, Goldwich A, Chauhan BK, Hudl K, Ohlmann AV, Zrenner E, Berger W, Cvekl A, Seeliger MW, Tamm ER (2005) Ectopic norrin induces growth of ocular capillaries and restores normal retinal angiogenesis in Norrie disease mutant mice. J Neurosci 25:1701 – 1710

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