- Email: [email protected]
Switching on the Lights: The Use of Optogenetics to Advance Retinal Gene Therapy
© The American Society of Gene & Cell Therapy
commentary blood products—and even terminally differentiated nondividing cell types—are likely to tolerate relatively high levels of genetic mutation in parental iPSCs with out significant oncogenic risk for a patient. Regeneration of tissues through trans plantation of lineage-directed stem and progenitor cells may take a little longer to establish. References
1. Takahashi, K and Yamanaka, S (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676. 2. Yu, J, Vodyanik, MA, Smuga-Otto, K, AntosiewiczBourget, J, Frane, JL, Tian, S et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920. 3. Park, IH, Arora, N, Huo, H, Maherali, N, Ahfeldt, T, Shimamura, A et al. (2008). Disease-specific induced pluripotent stem cells. Cell 134: 877–886. 4. Hanna, J, Wernig, M, Markoulaki, S, Sun, CW, Meissner, A, Cassady, JP et al. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318: 1920–1923. 5. Raya, A, Rodriguez-Piza, I, Guenechea, G, Vassena, R, Navarro, S, Barrero, MJ et al. (2009). Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature 460: 53–59. 6. Naldini, L (2011). Ex vivo gene transfer and correction for cell-based therapies. Nat Rev Genet 12: 301–315. 7. Mitchell, RS, Beitzel, BF, Schroder, AR, Shinn, P, Chen, H, Berry, CC et al. (2004). Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol 2: E234. 8. Winkler, T, Cantilena, A, Metais, JY, Xu, X, Nguyen, AD, Borate, B et al. (2010). No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells. Stem Cells 28: 687–694. 9. Kane, NM, Nowrouzi, A, Mukherjee, S, Blundell, MP, Greig, JA, Lee, WK et al. (2010). Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors. Mol Ther 18: 2139–2145. 10. Somers, A, Jean, JC, Sommer, CA, Omari, A, Ford, CC, Mills, JA et al. (2010). Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells 28: 1728–1740. 11. Kustikova, OS, Geiger, H, Li, Z, Brugman, MH, Chambers, SM, Shaw, CA et al. (2007). Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways. Blood 109: 1897–1907. 12. Warren, L, Manos, PD, Ahfeldt, T, Loh, YH, Li, H, Lau, F et al. (2010). Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7: 618–630. 13. Yu, J, Hu, K, Smuga-Otto, K, Tian, S, Stewart, R, Slukvin, II et al. (2009). Human induced pluripotent stem cells free of vector and transgene sequences. Science 324: 797–801. 14. Zhou, H, Wu, S, Joo, JY, Zhu, S, Han, DW, Lin, T et al. (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4: 381–384. 15. Papapetrou, EP, Lee, G, Malani, N, Setty, M, Riviere, I, Tirunagari, LM et al. (2011). Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol 29: 73–78. 16. Shi, Y, Do, JT, Desponts, C, Hahm, HS, Scholer, HR and Ding, S (2008). A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2: 525–528. 17. Huangfu, D, Maehr, R, Guo, W, Eijkelenboom, A, Snitow, M, Chen, AE et al. (2008). Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol 26: 795–797. 18. Nokye-Danso, F, Trivedi, CM, Juhr, D, Gupta, M, Cui,
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19.
20.
21.
22.
23.
24.
25.
Z, Tian, Y et al. (2011). Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8: 376–388. Li, H, Collado, M, Villasante, A, Strati, K, Ortega, S, Canamero, M et al. (2009). The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460: 1136–1139. Hong, H, Takahashi, K, Ichisaka, T, Aoi, T, Kanagawa, O, Nakagawa, M et al. (2009). Suppression of induced pluripotent stem cell generation by the p53‑p21 pathway. Nature 460: 1132–1135. Gore, A, Li, Z, Fung, HL, Young, JE, Agarwal, S, Antosiewicz-Bourget, J et al. (2011). Somatic coding mutations in human induced pluripotent stem cells. Nature 471: 63–67. Hussein, SM, Batada, NN, Vuoristo, S, Ching, RW, Autio, R, Narva, E et al. (2011). Copy number variation and selection during reprogramming to pluripotency. Nature 471: 58–62. Laurent, LC, Ulitsky, I, Slavin, I, Tran, H, Schork, A, Morey, R et al. (2011). Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell 8: 106–118. Howden, SE, Gore, A, Li, Z, Fung, HL, Nisler, BS, Nie, J et al. (2011). Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci USA 108: 6537–6542. Draper, JS, Moore, HD, Ruban, LN, Gokhale, PJ and Andrews PW (2004). Culture and characterization of human embryonic stem cells. Stem Cells Dev 13: 325–336.
26. Baker, DE, Harrison, NJ, Maltby, E, Smith, K, Moore, HD, Shaw, PJ et al. (2007). Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat Biotechnol 25: 207–215. 27. Mayshar, Y, Ben-David, U, Lavon, N, Biancotti, JC, Yakir, B, Clark, AT et al. (2010). Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7: 521–531. 28. Novak, A, Shtrichman, R, Germanguz, I, Segev, H, Zeevi-Levin, N, Fishman, B et al. (2010). Enhanced reprogramming and cardiac differentiation of human keratinocytes derived from plucked hair follicles, using a single excisable lentivirus. Cell Reprogram 12: 665–678. 29. Seki, T, Yuasa, S, Oda, M, Egashira, T, Yae, K, Kusumoto, D et al. (2010). Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7: 11–14. 30. Kunisato, A, Wakatsuki, M, Shinba, H, Ota, T, Ishida, I and Nagao, K (2011). Direct generation of induced pluripotent stem cells from human nonmobilized blood. Stem Cells Dev 20: 159–168. 31. Staerk, J, Dawlaty, MM, Gao, Q, Maetzel, D, Hanna, J, Sommer, CA et al. (2010). Reprogramming of human peripheral blood cells to induced pluripotent stem cells. Cell Stem Cell 7: 20–24. 32. Loh, YH, Hartung, O, Li, H, Guo, C, Sahalie, JM, Manos, PD et al. (2010). Reprogramming of T cells from human peripheral blood. Cell Stem Cell 7: 15–19. 33. Lister, R, Pelizzola, M, Kida, YS, Hawkins, RD, Nery, JR, Hon, G et al. (2011). Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471: 68–73.
See page 1212
Switching on the Lights: The Use of Optogenetics to Advance Retinal Gene Therapy Therese Cronin1 and Jean Bennett1 doi:10.1038/mt.2011.115
P
hotoreceptors carry a high number of mitochondria necessary to maintain their high metabolic rate. This allows them to respond rapidly to small changes in illumination. However, the metabolically demanding role of light reception appears to render photoreceptors particularly vulnerable to mutations; this is manifested in the many clinical forms of hereditary retinal degeneration (RD).1 Remarkably,
1 FM Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, USA Correspondence: Therese Cronin, FM Kirby Center for Molecular Ophthalmology, University of Pennsylvania, 309C Stellar-Chance Labs, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected]
in many cases the remainder of the retinal circuitry remains capable of firing despite the death of the photoreceptor cells.2,3 In this issue of Molecular Therapy, Caporale et al. exploit this residual capacity for sig nal transmission so as to endow retinal ganglion cells with the ability to take over the task of light detection from the photo receptors in conditions of RD.4 The retina is a thin sheet of photosensi tive neural tissue that lines the inside of the eyeball. It comprises three distinct layers of cells that vertically transmit the information carried by photons from the light-sensitive rod and cone photoreceptors via bipolar cells to the inner ganglion cells. Individuals with RD are progressively losing their rods and cones and face the prognosis of blind ness. Many of these patients cannot hope to benefit from the ongoing clinical trials for www.moleculartherapy.org vol. 19 no. 7 july 2011
© The American Society of Gene & Cell Therapy
commentary
Figure 1 LiGluR6-mediated therapeutic strategy in the retina. (a) Light signal transmission through the healthy retina. Rods and cones of the photoreceptor cell layer are shown at the top receiving photons. Signals are transmitted by specific cells in the bipolar layer (RBCs or OFF-BCs, depending on the wavelength and intensity of light) to ganglion cells at the bottom. (b) Light signal transmission through a degenerated retina expressing LiGluR6. The photons of light trigger LiGluR6 via activation of maleimide‑azobenzene‑glutamate (MAG) and lead to opening of the channel pore, bypassing the degenerated photoreceptors and the bipolar cell layer. (c) Detailed view of LiGluR6. MAG binding under 380-nm light activates the receptor and opens its cation-selective channel, resulting in membrane depolarization. a, azobenzene; g, glutamate; LBD, ligand-binding domain; OFF-BC, OFF-bipolar cell; RBC, retinal bipolar cell.
gene replacement or pharmaceutical thera pies, and a restoration of light sensitivity to the retina in its degenerated state may be the only possible treatment. The work by Caporale et al. was enabled by the growing field of optogenetics, which involves the heterologous expression of light-sensitive channels or receptors in a neuron, allow ing control of neuronal firing by an external light impulse.5 When applied to retinal gene therapy, these tools allow non-light-sensitive retinal cells to fire directly in response to light without the need to be triggered by Molecular Therapy vol. 19 no. 7 july 2011
the photoreceptor cells.3 These latest results from the Flannery laboratory4 support the potential of optogenetics to address the am bitious goal of vision restoration. Several groups in retinal research have used optogenetic tools from nature, such as channelrhodopsin2 (ChR2; ref. 3) and halorhodopsin (ref. 6). By contrast, Caporale et al.4 made use of an artifi cial optical switch that was engineered to generate large currents in neuronal cells.7 The switch, developed by Volgraf et al., is based on the ionotropic glutamate receptor
(iGluR) family.8 In addition to mediating the principal excitatory currents of the central nervous system, the iGluR fam ily carries a structurally ubiquitous ligandbinding domain that serves as a suitable template to achieve generic cell signaling. To the iGluR ligand-binding domain is incorporated a chemical called azoben zene which also serves as the carrier for the ligand glutamate; the entire moiety is termed maleimide‑azobenzene‑glutamate (MAG; see Figure 1). The light-dependent mechanics of the switch is achieved by the light-sensitive azobenzene, which undergoes isomerization from a trans to cis configuration in response to short wave lengths of light (≈380 nm). This azoben zene isomerization induces the molecular motion that allows glutamate (or its ana logs) to be drawn into the pore of the re ceptor and the cation channel to open. This is the trigger for membrane depolarization, the necessary step for the neuron to fire. The geometry is reversed and the channel closed under longer-wavelength light (≈500 nm) and in conditions of darkness. Caporale et al. applied this light-dependent iGluR6 prototype (LiGluR6) to the gan glion cell and demonstrated its potential as a platform for further engineering to meet the goal of reversal of blindness. To accomplish their results, the authors achieved a high level of ganglion cell‑spe cific LiGluR6 expression in the retinas of mice with retinal degeneration (rd1 mice). Tested ex vivo, electrical recordings show the retinas to be responsive to 380-nm light only when exposed to MAG, demonstrat ing the tight control of the switch. In vivo, photoresponses transmitted to the cortex were correlated in the behavior of entrained rd1 mice in a water-maze test. However, it is the restoration of the pupillary light re flex that sets this report apart from earlier studies of retinal optogenetic therapies. The pupillary light reflex refers to the contraction of the pupil as an involuntary response to light. This truly objective mea sure of non-image-forming photoresponse is mediated not only by the photoreceptors but also by a small subset of melanopsinexpressing ganglion cells.9,10 The authors selected the ideal mouse model in which to test this response: triple-knockout mice that lack melanopsin in addition to essen tial components of the phototransduction machinery and therefore do not show a 1191
© The American Society of Gene & Cell Therapy
commentary pupillary light reflex.11 Caporale and col leagues show that the reflex is restored in the mutant mice if their ganglion cells ex press LiGluR6 and they have been treated with MAG. It is important to note that targeting the ganglion cells may not take full ad vantage of the signal-processing power of the inner retina, where a lateral as well as a vertical transmission of information oc curs.12 Much of this processing takes place in the interneuron retinal bipolar cell layer before the light signal reaches the ganglion cells. Skipping the bipolar cells sacrifices to some extent two of their im portant processing roles: (i) discrimina tion of an object darker than background as compared with an object lighter than background and (ii) detection of the edges of an object whereby the center of the field is contrasted to an inhibitory surround. The absence of such processing would in theory lead to loss of resolution and noise reduction. A more immediate obstacle to be over come for clinical application of this strate gy is the requirement that a sufficient level of the photoswitch MAG be delivered to meet the demands of newly turned-over iGluR6 channels. Encapsulated cell thera py may offer the best solution by allowing long-term administration of the glutamate analog in the eye.13 With this technology still under development, its incorporation may slow the progress of the LiGluR6 op tical switch toward the clinic. Nonetheless, LiGluR6 has a significant advantage over naturally occurring optical switches such as ChR2. The main deficiency of ChR2 is its high level of desensitization, which reduces the overall current that can be achieved.5,14,15 In the future, channel modifications may improve such limita tions; in the meantime the LiGluR6‑MAG channel offers a higher level of channel conductance and can therefore achieve more adequate photon capture.
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Mammalian opsin is a tough act to fol low for those who endeavor to engineer photosensitive channels. For optogenetic retinal therapies to be translated to the clinic, specific hurdles must be overcome. Among these, the channels should be made receptive to more physiologically useful wavelengths, the efficiency of gene transfer to primate inner retinal cells needs to be im proved, and the retinal cells that express the channel must be carefully selected so as to optimize visual processing while occupying regions of intact retinal circuitry. Alternative strategies aimed at restor ing the visual function lost in RD are being investigated, and these too have their own hurdles. Retinal regeneration through the manipulation of stem cells combined with tissue engineering has been the focus of intense research efforts and yielded some recent successes.16 However, the molecular mechanisms needed for appropriate stem cell differentiation remain largely unknown, as are the requirements for successful inte gration and survival of the derived cells fol lowing transplantation. The microelectronic retinal implant has also shown much prom ise since clinical trials commenced with the epiretinal chip (SecondSight) in February 2002 (ref. 17). The progress of these chips to full clinical use remains limited until im proved resolution and full biocompatibility can be achieved. As for the optogenetic approach, all these strategies require that layers of the in ner retina and associated cell types maintain their architecture for a period of time follow ing photoreceptor degeneration. Although this is broadly true in many instances of RD, it may nonetheless be of benefit to promote the parallel development of neurotrophic therapies that may optimally support the health of inner retinal cells. The study de scribed here reminds us that if creative but methodical approaches are used to test strat egies for vision restoration in advanced RD, the obstacles to the clinic will be overcome.
References
1. Punzo, C, Kornacker, K and Cepko, CL (2009). Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa. Nat Neurosci 12: 44–52. 2. Mazzoni, F, Novelli, E and Strettoi, E (2008). Retinal ganglion cells survive and maintain normal dendritic morphology in a mouse model of inherited photoreceptor degeneration. J Neurosci 28: 14282–14292. 3. Lagali, PS, Balya, D, Awatramani, GB, Münch, TA, Kim, DS, Busskamp, V et al. (2008). Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat Neurosci 11: 667–675. 4. Caporale, N, Kolstad, KD, Lee, T, Tochitsky, I, Dalkara, D, Trauner, D et al. (2011). LiGluR restores visual responses in rodent models of inherited blindness. Mol Ther 19: 1212–1219. 5. Nagel, G, Szellas, T, Huhn, W, Kateriya, S, Adeishvili, N, Berthold, P et al. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci USA 100: 13940–13945. 6. Busskamp, V, Duebel, J, Balya, D, Fradot, M, Viney, TJ, Siegert, S et al. (2010). Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa. Science 329: 413–417. 7. Szobota, S, Gorostiza, P, Del Bene, F, Wyart, C, Fortin, DL, Kolstad, KD et al. (2007). Remote control of neuronal activity with a light-gated glutamate receptor. Neuron 54: 535–545. 8. Volgraf, M, Gorostiza, P, Numano, R, Kramer, RH, Isacoff, EY and Trauner, D. (2006). Allosteric control of an ionotropic glutamate receptor with an optical switch. Nat Chem Biol 2: 47–52. 9. Lucas, RJ, Hattar, S, Takao, M, Berson, DM, Foster, RG and Yau, K-W (2003). Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice. Science 299: 245–247. 10. Tsujimura, S, Ukai, K, Ohama, D, Nuruki, A and Yunokuchi, K (2010). Contribution of human melanopsin retinal ganglion cells to steady-state pupil responses. Proc Biol Sci 277: 2485–2492. 11. Hattar, S, Lucas, RJ, Mrosovsky, N, Thompson, S, Douglas, RH, Hankins, MW et al. (2003). Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424: 76–81. 12. Lagali, PS, Balya, D, Awatramani, GB, Münch, TA, Kim, DS, Busskamp, V et al. (2008). Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat Neurosci 11: 667–675. 13. Tao, W (2006). Application of encapsulated cell technology for retinal degenerative diseases. Expert Opin Biol Ther 6: 717–726. 14. Ishizuka, T, Kakuda, M, Araki, R and Yawo, H (2006). Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels. Neurosci Res 54: 85–94. 15. Lin, JY, Lin, MZ, Steinbach, P and Tsien, RY (2009). Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys J 96: 1803–1814. 16. Silva, GA, Silva, NM and Fortunato, TM (2011). Stem cell and tissue engineering therapies for ocular regeneration. Curr Stem Cell Res Ther; e-pub ahead of print 11 April 2011. 17. Chader, GJ, Weiland, J and Humayun, MS (2009). Artificial vision: needs, functioning, and testing of a retinal electronic prosthesis. Prog Brain Res 175: 317–332.
www.moleculartherapy.org vol. 19 no. 7 july 2011
commentary blood products—and even terminally differentiated nondividing cell types—are likely to tolerate relatively high levels of genetic mutation in parental iPSCs with out significant oncogenic risk for a patient. Regeneration of tissues through trans plantation of lineage-directed stem and progenitor cells may take a little longer to establish. References
1. Takahashi, K and Yamanaka, S (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676. 2. Yu, J, Vodyanik, MA, Smuga-Otto, K, AntosiewiczBourget, J, Frane, JL, Tian, S et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920. 3. Park, IH, Arora, N, Huo, H, Maherali, N, Ahfeldt, T, Shimamura, A et al. (2008). Disease-specific induced pluripotent stem cells. Cell 134: 877–886. 4. Hanna, J, Wernig, M, Markoulaki, S, Sun, CW, Meissner, A, Cassady, JP et al. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318: 1920–1923. 5. Raya, A, Rodriguez-Piza, I, Guenechea, G, Vassena, R, Navarro, S, Barrero, MJ et al. (2009). Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature 460: 53–59. 6. Naldini, L (2011). Ex vivo gene transfer and correction for cell-based therapies. Nat Rev Genet 12: 301–315. 7. Mitchell, RS, Beitzel, BF, Schroder, AR, Shinn, P, Chen, H, Berry, CC et al. (2004). Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol 2: E234. 8. Winkler, T, Cantilena, A, Metais, JY, Xu, X, Nguyen, AD, Borate, B et al. (2010). No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells. Stem Cells 28: 687–694. 9. Kane, NM, Nowrouzi, A, Mukherjee, S, Blundell, MP, Greig, JA, Lee, WK et al. (2010). Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors. Mol Ther 18: 2139–2145. 10. Somers, A, Jean, JC, Sommer, CA, Omari, A, Ford, CC, Mills, JA et al. (2010). Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells 28: 1728–1740. 11. Kustikova, OS, Geiger, H, Li, Z, Brugman, MH, Chambers, SM, Shaw, CA et al. (2007). Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways. Blood 109: 1897–1907. 12. Warren, L, Manos, PD, Ahfeldt, T, Loh, YH, Li, H, Lau, F et al. (2010). Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7: 618–630. 13. Yu, J, Hu, K, Smuga-Otto, K, Tian, S, Stewart, R, Slukvin, II et al. (2009). Human induced pluripotent stem cells free of vector and transgene sequences. Science 324: 797–801. 14. Zhou, H, Wu, S, Joo, JY, Zhu, S, Han, DW, Lin, T et al. (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4: 381–384. 15. Papapetrou, EP, Lee, G, Malani, N, Setty, M, Riviere, I, Tirunagari, LM et al. (2011). Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol 29: 73–78. 16. Shi, Y, Do, JT, Desponts, C, Hahm, HS, Scholer, HR and Ding, S (2008). A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2: 525–528. 17. Huangfu, D, Maehr, R, Guo, W, Eijkelenboom, A, Snitow, M, Chen, AE et al. (2008). Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol 26: 795–797. 18. Nokye-Danso, F, Trivedi, CM, Juhr, D, Gupta, M, Cui,
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19.
20.
21.
22.
23.
24.
25.
Z, Tian, Y et al. (2011). Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8: 376–388. Li, H, Collado, M, Villasante, A, Strati, K, Ortega, S, Canamero, M et al. (2009). The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460: 1136–1139. Hong, H, Takahashi, K, Ichisaka, T, Aoi, T, Kanagawa, O, Nakagawa, M et al. (2009). Suppression of induced pluripotent stem cell generation by the p53‑p21 pathway. Nature 460: 1132–1135. Gore, A, Li, Z, Fung, HL, Young, JE, Agarwal, S, Antosiewicz-Bourget, J et al. (2011). Somatic coding mutations in human induced pluripotent stem cells. Nature 471: 63–67. Hussein, SM, Batada, NN, Vuoristo, S, Ching, RW, Autio, R, Narva, E et al. (2011). Copy number variation and selection during reprogramming to pluripotency. Nature 471: 58–62. Laurent, LC, Ulitsky, I, Slavin, I, Tran, H, Schork, A, Morey, R et al. (2011). Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell 8: 106–118. Howden, SE, Gore, A, Li, Z, Fung, HL, Nisler, BS, Nie, J et al. (2011). Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci USA 108: 6537–6542. Draper, JS, Moore, HD, Ruban, LN, Gokhale, PJ and Andrews PW (2004). Culture and characterization of human embryonic stem cells. Stem Cells Dev 13: 325–336.
26. Baker, DE, Harrison, NJ, Maltby, E, Smith, K, Moore, HD, Shaw, PJ et al. (2007). Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat Biotechnol 25: 207–215. 27. Mayshar, Y, Ben-David, U, Lavon, N, Biancotti, JC, Yakir, B, Clark, AT et al. (2010). Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7: 521–531. 28. Novak, A, Shtrichman, R, Germanguz, I, Segev, H, Zeevi-Levin, N, Fishman, B et al. (2010). Enhanced reprogramming and cardiac differentiation of human keratinocytes derived from plucked hair follicles, using a single excisable lentivirus. Cell Reprogram 12: 665–678. 29. Seki, T, Yuasa, S, Oda, M, Egashira, T, Yae, K, Kusumoto, D et al. (2010). Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7: 11–14. 30. Kunisato, A, Wakatsuki, M, Shinba, H, Ota, T, Ishida, I and Nagao, K (2011). Direct generation of induced pluripotent stem cells from human nonmobilized blood. Stem Cells Dev 20: 159–168. 31. Staerk, J, Dawlaty, MM, Gao, Q, Maetzel, D, Hanna, J, Sommer, CA et al. (2010). Reprogramming of human peripheral blood cells to induced pluripotent stem cells. Cell Stem Cell 7: 20–24. 32. Loh, YH, Hartung, O, Li, H, Guo, C, Sahalie, JM, Manos, PD et al. (2010). Reprogramming of T cells from human peripheral blood. Cell Stem Cell 7: 15–19. 33. Lister, R, Pelizzola, M, Kida, YS, Hawkins, RD, Nery, JR, Hon, G et al. (2011). Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471: 68–73.
See page 1212
Switching on the Lights: The Use of Optogenetics to Advance Retinal Gene Therapy Therese Cronin1 and Jean Bennett1 doi:10.1038/mt.2011.115
P
hotoreceptors carry a high number of mitochondria necessary to maintain their high metabolic rate. This allows them to respond rapidly to small changes in illumination. However, the metabolically demanding role of light reception appears to render photoreceptors particularly vulnerable to mutations; this is manifested in the many clinical forms of hereditary retinal degeneration (RD).1 Remarkably,
1 FM Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, USA Correspondence: Therese Cronin, FM Kirby Center for Molecular Ophthalmology, University of Pennsylvania, 309C Stellar-Chance Labs, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected]
in many cases the remainder of the retinal circuitry remains capable of firing despite the death of the photoreceptor cells.2,3 In this issue of Molecular Therapy, Caporale et al. exploit this residual capacity for sig nal transmission so as to endow retinal ganglion cells with the ability to take over the task of light detection from the photo receptors in conditions of RD.4 The retina is a thin sheet of photosensi tive neural tissue that lines the inside of the eyeball. It comprises three distinct layers of cells that vertically transmit the information carried by photons from the light-sensitive rod and cone photoreceptors via bipolar cells to the inner ganglion cells. Individuals with RD are progressively losing their rods and cones and face the prognosis of blind ness. Many of these patients cannot hope to benefit from the ongoing clinical trials for www.moleculartherapy.org vol. 19 no. 7 july 2011
© The American Society of Gene & Cell Therapy
commentary
Figure 1 LiGluR6-mediated therapeutic strategy in the retina. (a) Light signal transmission through the healthy retina. Rods and cones of the photoreceptor cell layer are shown at the top receiving photons. Signals are transmitted by specific cells in the bipolar layer (RBCs or OFF-BCs, depending on the wavelength and intensity of light) to ganglion cells at the bottom. (b) Light signal transmission through a degenerated retina expressing LiGluR6. The photons of light trigger LiGluR6 via activation of maleimide‑azobenzene‑glutamate (MAG) and lead to opening of the channel pore, bypassing the degenerated photoreceptors and the bipolar cell layer. (c) Detailed view of LiGluR6. MAG binding under 380-nm light activates the receptor and opens its cation-selective channel, resulting in membrane depolarization. a, azobenzene; g, glutamate; LBD, ligand-binding domain; OFF-BC, OFF-bipolar cell; RBC, retinal bipolar cell.
gene replacement or pharmaceutical thera pies, and a restoration of light sensitivity to the retina in its degenerated state may be the only possible treatment. The work by Caporale et al. was enabled by the growing field of optogenetics, which involves the heterologous expression of light-sensitive channels or receptors in a neuron, allow ing control of neuronal firing by an external light impulse.5 When applied to retinal gene therapy, these tools allow non-light-sensitive retinal cells to fire directly in response to light without the need to be triggered by Molecular Therapy vol. 19 no. 7 july 2011
the photoreceptor cells.3 These latest results from the Flannery laboratory4 support the potential of optogenetics to address the am bitious goal of vision restoration. Several groups in retinal research have used optogenetic tools from nature, such as channelrhodopsin2 (ChR2; ref. 3) and halorhodopsin (ref. 6). By contrast, Caporale et al.4 made use of an artifi cial optical switch that was engineered to generate large currents in neuronal cells.7 The switch, developed by Volgraf et al., is based on the ionotropic glutamate receptor
(iGluR) family.8 In addition to mediating the principal excitatory currents of the central nervous system, the iGluR fam ily carries a structurally ubiquitous ligandbinding domain that serves as a suitable template to achieve generic cell signaling. To the iGluR ligand-binding domain is incorporated a chemical called azoben zene which also serves as the carrier for the ligand glutamate; the entire moiety is termed maleimide‑azobenzene‑glutamate (MAG; see Figure 1). The light-dependent mechanics of the switch is achieved by the light-sensitive azobenzene, which undergoes isomerization from a trans to cis configuration in response to short wave lengths of light (≈380 nm). This azoben zene isomerization induces the molecular motion that allows glutamate (or its ana logs) to be drawn into the pore of the re ceptor and the cation channel to open. This is the trigger for membrane depolarization, the necessary step for the neuron to fire. The geometry is reversed and the channel closed under longer-wavelength light (≈500 nm) and in conditions of darkness. Caporale et al. applied this light-dependent iGluR6 prototype (LiGluR6) to the gan glion cell and demonstrated its potential as a platform for further engineering to meet the goal of reversal of blindness. To accomplish their results, the authors achieved a high level of ganglion cell‑spe cific LiGluR6 expression in the retinas of mice with retinal degeneration (rd1 mice). Tested ex vivo, electrical recordings show the retinas to be responsive to 380-nm light only when exposed to MAG, demonstrat ing the tight control of the switch. In vivo, photoresponses transmitted to the cortex were correlated in the behavior of entrained rd1 mice in a water-maze test. However, it is the restoration of the pupillary light re flex that sets this report apart from earlier studies of retinal optogenetic therapies. The pupillary light reflex refers to the contraction of the pupil as an involuntary response to light. This truly objective mea sure of non-image-forming photoresponse is mediated not only by the photoreceptors but also by a small subset of melanopsinexpressing ganglion cells.9,10 The authors selected the ideal mouse model in which to test this response: triple-knockout mice that lack melanopsin in addition to essen tial components of the phototransduction machinery and therefore do not show a 1191
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commentary pupillary light reflex.11 Caporale and col leagues show that the reflex is restored in the mutant mice if their ganglion cells ex press LiGluR6 and they have been treated with MAG. It is important to note that targeting the ganglion cells may not take full ad vantage of the signal-processing power of the inner retina, where a lateral as well as a vertical transmission of information oc curs.12 Much of this processing takes place in the interneuron retinal bipolar cell layer before the light signal reaches the ganglion cells. Skipping the bipolar cells sacrifices to some extent two of their im portant processing roles: (i) discrimina tion of an object darker than background as compared with an object lighter than background and (ii) detection of the edges of an object whereby the center of the field is contrasted to an inhibitory surround. The absence of such processing would in theory lead to loss of resolution and noise reduction. A more immediate obstacle to be over come for clinical application of this strate gy is the requirement that a sufficient level of the photoswitch MAG be delivered to meet the demands of newly turned-over iGluR6 channels. Encapsulated cell thera py may offer the best solution by allowing long-term administration of the glutamate analog in the eye.13 With this technology still under development, its incorporation may slow the progress of the LiGluR6 op tical switch toward the clinic. Nonetheless, LiGluR6 has a significant advantage over naturally occurring optical switches such as ChR2. The main deficiency of ChR2 is its high level of desensitization, which reduces the overall current that can be achieved.5,14,15 In the future, channel modifications may improve such limita tions; in the meantime the LiGluR6‑MAG channel offers a higher level of channel conductance and can therefore achieve more adequate photon capture.
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Mammalian opsin is a tough act to fol low for those who endeavor to engineer photosensitive channels. For optogenetic retinal therapies to be translated to the clinic, specific hurdles must be overcome. Among these, the channels should be made receptive to more physiologically useful wavelengths, the efficiency of gene transfer to primate inner retinal cells needs to be im proved, and the retinal cells that express the channel must be carefully selected so as to optimize visual processing while occupying regions of intact retinal circuitry. Alternative strategies aimed at restor ing the visual function lost in RD are being investigated, and these too have their own hurdles. Retinal regeneration through the manipulation of stem cells combined with tissue engineering has been the focus of intense research efforts and yielded some recent successes.16 However, the molecular mechanisms needed for appropriate stem cell differentiation remain largely unknown, as are the requirements for successful inte gration and survival of the derived cells fol lowing transplantation. The microelectronic retinal implant has also shown much prom ise since clinical trials commenced with the epiretinal chip (SecondSight) in February 2002 (ref. 17). The progress of these chips to full clinical use remains limited until im proved resolution and full biocompatibility can be achieved. As for the optogenetic approach, all these strategies require that layers of the in ner retina and associated cell types maintain their architecture for a period of time follow ing photoreceptor degeneration. Although this is broadly true in many instances of RD, it may nonetheless be of benefit to promote the parallel development of neurotrophic therapies that may optimally support the health of inner retinal cells. The study de scribed here reminds us that if creative but methodical approaches are used to test strat egies for vision restoration in advanced RD, the obstacles to the clinic will be overcome.
References
1. Punzo, C, Kornacker, K and Cepko, CL (2009). Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa. Nat Neurosci 12: 44–52. 2. Mazzoni, F, Novelli, E and Strettoi, E (2008). Retinal ganglion cells survive and maintain normal dendritic morphology in a mouse model of inherited photoreceptor degeneration. J Neurosci 28: 14282–14292. 3. Lagali, PS, Balya, D, Awatramani, GB, Münch, TA, Kim, DS, Busskamp, V et al. (2008). Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat Neurosci 11: 667–675. 4. Caporale, N, Kolstad, KD, Lee, T, Tochitsky, I, Dalkara, D, Trauner, D et al. (2011). LiGluR restores visual responses in rodent models of inherited blindness. Mol Ther 19: 1212–1219. 5. Nagel, G, Szellas, T, Huhn, W, Kateriya, S, Adeishvili, N, Berthold, P et al. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci USA 100: 13940–13945. 6. Busskamp, V, Duebel, J, Balya, D, Fradot, M, Viney, TJ, Siegert, S et al. (2010). Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa. Science 329: 413–417. 7. Szobota, S, Gorostiza, P, Del Bene, F, Wyart, C, Fortin, DL, Kolstad, KD et al. (2007). Remote control of neuronal activity with a light-gated glutamate receptor. Neuron 54: 535–545. 8. Volgraf, M, Gorostiza, P, Numano, R, Kramer, RH, Isacoff, EY and Trauner, D. (2006). Allosteric control of an ionotropic glutamate receptor with an optical switch. Nat Chem Biol 2: 47–52. 9. Lucas, RJ, Hattar, S, Takao, M, Berson, DM, Foster, RG and Yau, K-W (2003). Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice. Science 299: 245–247. 10. Tsujimura, S, Ukai, K, Ohama, D, Nuruki, A and Yunokuchi, K (2010). Contribution of human melanopsin retinal ganglion cells to steady-state pupil responses. Proc Biol Sci 277: 2485–2492. 11. Hattar, S, Lucas, RJ, Mrosovsky, N, Thompson, S, Douglas, RH, Hankins, MW et al. (2003). Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424: 76–81. 12. Lagali, PS, Balya, D, Awatramani, GB, Münch, TA, Kim, DS, Busskamp, V et al. (2008). Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat Neurosci 11: 667–675. 13. Tao, W (2006). Application of encapsulated cell technology for retinal degenerative diseases. Expert Opin Biol Ther 6: 717–726. 14. Ishizuka, T, Kakuda, M, Araki, R and Yawo, H (2006). Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels. Neurosci Res 54: 85–94. 15. Lin, JY, Lin, MZ, Steinbach, P and Tsien, RY (2009). Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys J 96: 1803–1814. 16. Silva, GA, Silva, NM and Fortunato, TM (2011). Stem cell and tissue engineering therapies for ocular regeneration. Curr Stem Cell Res Ther; e-pub ahead of print 11 April 2011. 17. Chader, GJ, Weiland, J and Humayun, MS (2009). Artificial vision: needs, functioning, and testing of a retinal electronic prosthesis. Prog Brain Res 175: 317–332.
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