the eye seems warranted. Current treatment modalities for ocular cancer involve surgery, radiation, chemotherapy, thermochemotherapy, immunotherapy, and photodynamic therapy. The success of these strategies is mixed and depends on the stage and type of cancer in question. These treatments are destructive and often come with severe side-effects. Gene therapy may lead to treatments which effectively target and neutralize malignant cells while preserving ocular function. Two promising strategies employ use of apoptotic-inducing molecules or nonnative suicide genes. Genes that induce apoptosis, such as p53, Bax, and caspase-3, are excellent candidates and their functions are beginning to be understood. By specifically targeting cancerous cells with viral vectors expressing proapoptotic factors, elimination of these cells while avoiding damage to functioning “bystander” tissue may be possible. Another approach to cancer treatment via gene therapy is to target the delivery or expression of molecules with proteolytic activity. Clearly this will require accurate targeting of the malignant cells or the effective control of expression of these toxic genes within the desired cells.

RETINOBLASTOMA GENE THERAPY CLINICAL TRIAL

Retinoblastoma is an intraocular tumor of the retina arising predominantly in pediatric populations and contributes to 6.1% of all cancers in children aged 0–4 years old. The US incidence rate for this age group is approximately 11.8 cases per million children, which correlates well with other reporting countries.30 The most common form of this cancer arises from a mutation in the RB1 gene, a factor responsible for cell cycle regulation, resulting in aggressive cellular growth that requires swift recognition and therapeutic intervention. Currently the most definitive means of treatment is enucleation; however eyes are now saved by a combination of chemotherapy, radiation, and laser treatments.

The herpes simplex virus synthesizes a form of Thymidine Kinase (TK), which differs significantly from the human form. Ganciclovir has been shown to bind specifically to viral TK, after which it is phosphorylated and competes with guanosine triphosphate binding. This action effectively impedes DNA synthesis and induces apoptosis via a p53mediated pathway. Thus, cells expressing the viral TK gene are susceptible to killing with ganciclovir, a drug that is innocuous to normal human cells. These data led researchers to investigate a combination therapy strategy for the treatment of various cancers, including retinoblastoma. After tumor shrinkage was observed in rodents receiving adenoviral-delivered herpes simplex thymidine kinase (Ad-TK) with systemic administration of ganciclovir, a human clinical trial was conducted to test whether this vector, when injected into the eyes of children with aggressive retinoblastoma, would reduce the size of tumors.

The phase I trial consisted of 8 children with active bilateral retinoblastoma. In addition, patients must have had the presence of vitreous seeds, had prior treatment failure, and had at least limited remaining visual acuity. The Ad-TK particles were delivered via transcorneal injection in order to minimize tumor spread through the needle tract. The vector was administered in a dose-escalating fashion with the lowest dose being 108 particles up to the highest dosage of 1011 particles. After 24 postinjection hours, patients began the first of 14 doses of ganciclovir (5 mg/kg) given at 12-hour intervals. If the retinoblastoma was considered to progress, these eyes were to be enucleated, and if no resolution of the tumor occurred with the absence of an adverse reaction, then multiple Ad-TK injections were considered.31

The results of this clinical trial were partially encouraging. Of the 8 patients enrolled in the study, 7 demonstrated some vitreous seed resolution. The other patient, who received the lowest dose of 108 particles, had tumor resolution around the injection site alone. Minor complications included mild inflammation, corneal edema, and increased intraocular pressure: some of these complications are believed to be the result of prior treatment attempts. There was no evidence of viral vector spread to other organs and viral shedding was not seen. The Ad-TK vector appeared safe but ineffective. All of the patients enrolled in the study did require enucleation at some point after treatment, with best

outcome persisting for 38 months postinjection.31 Whether this lack of efficacy was the result of the transgene or the vector employed remains unclear. Clearly, transgene expression from adenoviral vectors is limited in duration.

THE AdPEDF MACULAR DEGENERATION

CLINICAL TRIAL

AMD accounts for the majority of all blindness in people over the age of 50 in industrialized nations.32 Approximately 90% of vision loss associated with AMD is attributed to choroidal neovascularization (CNV),33 in which uncontrolled blood vessel growth leads to irreversible scarring and vision loss. Several genetic targets for AMD have been elucidated within the last several years, although VEGF and PEDF have been the most widely investigated to date.

The VGEF isoforms are members of the cysteine-knot growth factor family. VEGF-A is a potent signaling molecule capable of up-regulating migration and mitosis in endothelial cells as well as acting as a chemo­ attractant for macrophages and granulocytes. PEDF, expressed in the retinal pigment epithelium, is part of the serine protease inhibitor family of molecules. Several groups have shown that the overexpression of PEDF in eyes with CNV results in a significant decrease in neovascularization. In addition, exogenous introduction of PEDF was shown to inhibit laser-induced CNV effectively in various animal models.34,35

These data prompted the initiation of a phase I clinical trial of 28 patients to examine the potential of PEDF to treat CNV in patients with AMD. Patients enrolled were at least 50 years of age, had a neovascular AMD diagnosis with active CNV, and visual acuity of 20/200 or poorer. The study was performed in a dose-escalating fashion with doses ranging from 106 to 109.5 particles.36

The trial demonstrated that the intravitreal injection of AdPEDF was well tolerated. There were few complications as a result of the injection aside from mild inflammation, corneal edema, and elevated intraocular pressure in some of the patients, all of which were managed via topical medications. There was no evidence of systemic hematogenous vector spread and patients showed little sign of a systemic immune response to the drug. Although the study was primarily performed to test the safety of the vector, there were some signs of efficacy. Patients who received higher doses had no change in visual acuity as compared to low-dose patients whose acuity appeared to worsen over the course of the study. Vessel hyperpermeability appeared to resolve in some highdose patients over a 12-week period. Retinal appearance was improved with significantly less hyperfluorescence present in some patients evidenced by fluorescein angiography.36 The overall success of this trial has lead to the initiation of a second trial to examine the role of AdPEDF.11 on patients with early, less severe AMD.

GENE THERAPY FOR LEBER’S CONGENITAL AMAUROSIS TRIAL

LCA is an inherited blinding retinal disorder which presents typically within the first few months of postnatal life and for which there is no treatment.37 There are over a dozen known gene mutations that result in LCA; all forms produce a breakdown in the photo transduction cascade. Although the retina in these patients appears normal via fundus and histological examination, there is extreme retinal signaling dysfunction, as measured by electroretinogram (ERG). Current gene therapy trials (three are ongoing and another is planned) are targeted at replacing a defective RPE65 protein. The RPE65 protein is responsible for the conversion of all-trans-retinyl ester to 11-cis-retinol, a key stage of the visual pathway. The gene transfer vehicle being investigated is the type 2 AAV (AAV2). Recombinant AAV2 has been studied for efficacy in a variety of gene therapy models and has been shown to nontoxic and capable of long-term expression in nondividing cells. Its success for the treatment of LCA has been evaluated in both mouse and

Diseases Retinal in Mechanisms and Drugs • 4 section

• 41 chapterTherapy Gene Ocular

dog models of LCA disease. Treatment in the dog model has been documented to persist for over 3 years.38

One LCA human study is a combination phase I/II trial, to test both dose toxicity and efficacy in 12 patients. The second and fourth cohort of this trial will include children from 8 to 18 years of age. Participants will be placed into one of four groups and will receive one subretinal injection of the rAAV2-CB-hRPE65 vector at a concentration of either 1.8 × 1011 or 6 × 1011 viral particles. Efficacy will be graded by several parameters, including visual field and acuity changes as well as ERG testing, fundus photography, optical coherence tomography, and quality-of-life surveys.

Current animal data suggest that the overall tolerance to the drug will be well tolerated with little to no side-effects such as edema or intraocular pressure variation.39 Considering the choice of vector, AAV is not likely to be immunogenic. Based on the fact that LCA is a monogenic disease and the results documented in various animal models, the potential for visual improvement is quite promising.40,41 Although the incidence of LCA is rare – approximately 3 of every 100 000 births42

– the success of this treatment could improve the quality of life for a great deal of individuals worldwide as well as advancing the role of gene therapy as a treatment option.

SUMMARY AND KEYPOINTS: THE FUTURE OF GENE THERAPY

As of 2004 there were 613 approved gene therapy trials under way in the USA alone and that number will surely grow in the next several years.43 As research continues to elucidate the mysteries of the genome, transcriptome, and proteome, our understanding of more complex disease processes will improve. For gene therapy to realize its potential, several key features need to be addressed. Cell-specific targeting, inducible (and repressible) transgene expression, immunogenicity, and minimally invasive delivery techniques will be important features to study.44 Many advances in vector construction leading to safer more robust systems are under way. The ability to infect specific cell types is being investigated by pseudotyping lentiviral vectors with antibody fragments which bind very specific cellular receptors.45 Other modifications may eventually lead to site-specific integration, minimizing the risk of random gene silencing or activation, an inherit risk of integrating vectors. One group has demonstrated that, with the use of nonviral recombinase molecules derived from yeast, specific integration is feasible, albeit still in the early stages of discovery.46 While vectors will continue to improve and treatments become safer, public opinion and concerns will remain. Moral and ethical considerations will always be an important issue. The last two decades have demonstrated incredible progress – the future of gene therapy looks stronger than ever.

REFERENCES

1.Friedmann T. A brief history of gene therapy. Nat Genet 1992;2(2):93–98.

2.Sambrook J. Transformation by polyoma virus and simian virus 40. Adv Cancer Res 1972;16:141–180.

3.Sambrook J, Shatkin AJ. Polynucleotide ligase activity in cells infected with simian virus 40, polyoma virus, or vaccinia virus. J Virol 1969;4(5): 719–726.

4.Sambrook J, Westphal H, Srinivasan PR, et al. The integrated state of viral DNA in SV40-transformed cells. Proc Natl Acad Sci USA 1968;60(4): 1288–1295.

5.Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973;52(2):456–467.

6.Mulligan RC, Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci USA 1981;78:2072–2076.

7.Yamada M, Lewis JA, Grodzicker T, et al. Overproduction of the protein product of a nonselected foreign gene carried by an adenovirus vector. Proc Natl Acad Sci USA 1985;82(11):3567–3571.

8.Nakai H, Yant SR, Storm TA, et al. Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol 2001;75(15):6969–6976.

9.Gilboa E, Kolbe M, Noonan K, et al. Construction of a mammalian transducing vector from the genome of Moloney murine leukemia virus. J Virol 1982;44(3):845–851.

10.Feuerman MH, Davis BR, Pattengale PK, et al. Generation of a recombinant Moloney murine leukemia virus carrying the v-src gene of avian sarcoma virus: transformation in vitro and pathogenesis in vivo. J Virol 1985;54(3):804–816.

11.Miller AD, Jolly DJ, Friedmann T, et al. A transmissible retrovirus expressing human hypoxanthine phosphoribosyltransferase (HPRT): gene transfer into cells obtained from humans deficient in HPRT. Proc Natl Acad Sci USA 1983;80(15):4709–4713.

12.Miller AD, Eckner RJ, Jolly DJ, et al. Expression of a retrovirus encoding human HPRT in mice. Science 1984;225(4662):630–632.

13.Hlavaty J, Schittmayer M, Stracke A, et al. Effect of posttranscriptional regulatory elements on transgene expression and virus production in the context of retrovirus vectors. Virology 2005;341(1):1–11.

14.Van Maele B, De Rijck J, De Clercq E, et al. Impact of the central polypurine tract on the kinetics of human immunodeficiency virus type 1 vector transduction. J Virol 2003;77(8):4685–4694.

15.Loewen N, Leske DA, Cameron JD, et al. Long-term retinal transgene expression with FIV versus adenoviral vectors. Mol Vis 2004;10: 272–280.

16.Berg P, Baltimore D, Brenner S, et al. Summary statement of the Asilomar conference on recombinant DNA molecules. Proc Natl Acad Sci USA 1975;72(6):1981–1984.

17.Dickson D. NIH censure for Dr Martin Cline: tighter rules for future research plans. Nature 1981;291(5814):369.

18.Sun M. Martin Cline loses appeal on NIH grant. Science 1982;218(4567): 37.

19.Blaese RM, Culver KW, Miller AD, et al. T lymphocyte-directed gene therapy for ADASCID: initial trial results after 4 years. Science 1995;270(5235):475–480.

20.Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348(3):255–256.

21.Stolberg SG. The biotech death of Jesse Gelsinger. N Y Times Mag 1999;136–140, 149–150.

22.National Institutes of Health. Assessment of adenoviral vector safety and toxicity: report of the National Institutes of Health Recombinant DNA Advisory Committee. Hum Gene Ther 2002;13(1):3–13.

23.Bertling W, Hunger-Bertling K, Cline MJ. Intranuclear uptake and persistence of biologically active DNA after electroporation of mammalian cells. J Biochem Biophys Methods 1987;14(4):223–232.

24.Krotz F, Sohn HY, Gloe T, et al. Magnetofection potentiates gene delivery to cultured endothelial cells. J Vasc Res 2003;40(5):425–434.

25.Le Meur G, Stieger K, Smith AJ, et al. Restoration of vision in RPE65deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium. Gene Ther 2007;14(4):292–303.

26.O’Reilly M, Palfi A, Chadderton N, et al. RNA interference-mediated suppression and replacement of human rhodopsin in vivo. Am J Hum Genet 2007;81(1):127–135.

27.Klettner A, Roider J. Comparison of bevacizumab, ranibizumab, and pegaptanib in vitro: efficiency and possible additional pathways. Invest Ophthalmol Vis Sci 2008;49(10):4523–4527.

28.Sallinen H, Anttila M, Narvainen J, et al. Antiangiogenic gene therapy with soluble VEGFR-1, -2, and -3 reduces the growth of solid human ovarian carcinoma in mice. Mol Ther 2009;(in press).

29.Appukuttan B, McFarland TJ, Davies MH, et al. Identification of novel alternatively spliced isoforms of RTEF-1 within human ocular vascular endothelial cells and murine retina. Invest Ophthalmol Vis Sci 2007;48(8): 3775–3782.

30.Broaddus E, Topham A, Singh AD, et al. Incidence of retinoblastoma in the USA:1975–2004. Br J Ophthalmol 2009;93(1):21–23.

31.Chevez-Barrios P, Chintagumpala M, Mieler W, et al. Response of retinoblastoma with vitreous tumor seeding to adenovirus-mediated delivery of thymidine kinase followed by ganciclovir. J Clin Oncol 2005;23(31): 7927–7935.

32.Korner-Stiefbold U. Age-related macular degeneration (AMD) – therapeutic possibilities and new approaches. Ther Umsch 2001;58(1):28–35.

33.Polito A, Isola M, Lanzetta P, et al. The natural history of occult choroidal neovascularisation associated with age-related macular degeneration. A systematic review. Ann Acad Med Singapore 2006;35(3):145–150.

34.Duh EJ, Yang HS, Suzuma I, et al. Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth. Invest Ophthalmol Vis Sci 2002;43(3):821–829.

35.Mori K, Duh E, Gehlbach P, et al. Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization. J Cell Physiol 2001;188(2):253–263.

36.Campochiaro PA, Nguyen QD, Shah SM, et al. Adenoviral vector-delivered pigment epithelium-derived factor for neovascular age-related macular degeneration: results of a phase I clinical trial. Hum Gene Ther 2006;17(2):167–176.

37.Cremers FP, van den Hurk JA, et al. Molecular genetics of Leber congenital amaurosis. Hum Mol Genet 2002;11(10):1169–1176.

38.Narfstrom K, Vaegan T, Katz M, et al. Assessment of structure and function over a 3-year period after gene transfer in RPE65-/- dogs. Doc Ophthalmol 2005;111(1):39–48.

39.Acland GM, Aguirre GD, Bennett J, et al. Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. Mol Ther 2005;12(6):1072–1082.

40.Acland GM, Aguirre GD, Ray J, et al. Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 2001;28(1):92–95.

41.Narfstrom K, Katz ML, Bragadottir R, et al. Functional and structural recovery of the retina after gene therapy in the RPE65 null mutation dog. Invest Ophthalmol Vis Sci 2003;44(4):1663–1672.

42.Fazzi E, Signorini SG, Scelsa B, et al. Leber’s congenital amaurosis: an update. Eur J Paediatr Neurol 2003;7(1):13–22.

43.Cavazzana-Calvo M, Thrasher A, Mavilio F. The future of gene therapy. Nature 2004;427(6977):779–781.

44.McCain J. The future of gene therapy. Biotechnology Healthcare 2005;53–60.

45.Aires da Silva F, Costa MJ, Corte-Real S, et al. Cell type-specific targeting with sindbis pseudotyped lentiviral vectors displaying anti-CCR5 singlechain antibodies. Hum Gene Ther 2005;16(2):223–234.

46.Moldt B, Staunstrup NH, Jakobsen M, et al. Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase. BMC Biotechnol 2008;8:60.

Diseases Retinal in Mechanisms and Drugs • 4 section