Título provisional de la tesis

Morphological and electrophysiological characterization of optogenetic variants in vitro and in vivo using nanoparticles as vectors for purposes of visual restoration.

Resumen

Retinitis pigmentosa (RP) is defined as a heterogeneous group of inherited retinal disorders characterized by progressive degeneration of the photoreceptors with subsequent degeneration of the retinal pigment epithelium (RPE) (1-3). Many treatments have been carried out to cure or stop this degeneration, such as vitamin supplementation (4), cell transplantation (5) or gene therapy (6), but all of them just delay the degenerative process. The most significant advances in visual restoration have been made using retinal multi-electrode implants (7-8), but the vision achieved is limited at best and the cost excludes the general public from benefiting from this method.

Optogenetics, a method which uses targeted ectopic expression of light-activated proteins to control neural activity with temporal precision (9) has emerged as a promising alternative in visual rehabilitation. Studies have demonstrated that cell-specific targeted expression of the microbial opsin Channelrhodopsin-2 (ChR2) in the remaining neural tissue of degenerated retinae, such as the bipolar and ganglion cells, restore light sensitivity which is transmitted to higher visual centers driving light induced visual behavior (10-12). However, one shortcoming of these original studies was the high intensity of blue light required to stimulate ChR2 which can potentially cause photochemical mitochondrial damage through reactive oxygen species production (13).

Efficient delivery and expression of opsin genes is critical for achieving spatiotemporally-resolved cell type–specific manipulation. One method, popular because it allows for tight control over spatial localization of opsin expression, is through the use of viral vector targeting systems. Using this approach, an engineered virus containing an opsin gene driven by a specific promoter is injected into the brain region of interest. This method offers fast and robust expression. Various viral vectors such as lentivirus, adeno-associated virus, rabies virus, canine adenovirus, and herpes simplex virus-useful for different applications-have been used to introduce opsins to different systems, including mouse, rat, zebrafish, and primate models (14-15). However, the payloads (the length of genetic material a virus can carry) of these viruses are limited, thus limiting the size of the promoter and thereby reducing the diversity of cell types that can be specifically targeted with sufficient expression and can cause immunogenic responses.

Unlike the viral methods, electroporation (a physical transfection method that uses an electrical pulse to create temporary pores in cell membranes through which substances like nucleic acids can pass into cells) may deliver DNA of any size with large promoter segments to achieve high cellular specificity and does not have immunogenic responses. Electroporation also allows to inject numerous gene copies, but can cause a big reduction of the size of the eye called microphthalmia (16).

This thesis is based in the use of safer nanoparticles formulations to transfect the retina of RD10 mice (a widely used animal model for retinitis pigmentosa) and cortical cultures from Sprague dawley rat two improved optogenetic variants of ChR2, which are CatCh (17) and ChrimsonR (18). CatCh requires less blue-light intensity than ChR2 to induce spiking in neurons reducing any phototoxic effects, whereas ChrimsonR is a red-shifted channelrhodopsin with a peak spectrum of 590 nm that allows for deeper tissue penetration and independent optical excitation of distinct neural populations. In this thesis it will be a comparison of this transfection method with electroporation and viral vectors through cellular, electrophysiological and behavioral studies.

1. Marshall J, Heckenlively JR. Retinitis pigmentosa. Philadelphia: J.B. Lippincott Co; 1988:37-42.

2. Weleber R. Retinitis pigmentosa and allied disorders. In: Ryan S, Ogden T, Schachat A, eds. Retina. 2nd ed. St. Louis: Mosby-Year Book, Inc; 1994:334-40.

3. Berson E. Retinitis pigmentosa and allied diseases. In: Albert DM, Jakobied FA, eds. Principles and practice of ophthalmology: clinical practice, Vol 3, 2nd ed. Philadelphia: WB Saunders; 1994:1214.

4. Vitamin A and fish oils for retinitis pigmentosa.Rayapudi S, Schwartz SG, Wang X, Chavis P.Cochrane Database Syst Rev. 2013 Dec 19;(12)

5. Cortical visual functions can be preserved by subretinal RPE cell grafting in RCS rats. Girman S.V., Wang S., Lund R.D. Vis. Res. 2003;43:1817–1827.

6. CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa. Wu WH, Tsai YT, Justus S, Lee TT, Zhang L, Lin CS, Bassuk AG, Mahajan VB, Tsang SH. Mol Ther. 2016 Aug;24(8):1388-94.

7. Blind subjects implanted with the Argus II retinal prosthesis are able to improve performance in a spatial-motor task. Ahuja AK, Dorn JD, Caspi A, McMahon MJ, Dagnelie G, Dacruz L, Stanga P, Humayun MS, Greenberg RJ; Argus II Study Group. Br J Ophthalmol. 2011 Apr;95(4):539-43.

8. Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS. Stingl K, Bartz-Schmidt KU, Besch D, Braun A, Bruckmann A, Gekeler F, Greppmaier U, Hipp S, Hörtdörfer G, Kernstock C, Koitschev A, Kusnyerik A, Sachs H, Schatz A, Stingl KT, Peters T, Wilhelm B, Zrenner E.Proc Biol Sci. 2013 Feb 20;280(1757):20130077

9. Millisecond-timescale, genetically targeted optical control of neural activity. E.S. Boyden, F. Zhang, E. Bamberg, G. Nagel, K. Deisseroth. Nat. Neurosci. 8 (9) (2005) 1263–1268.

10. Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Lagali PS, Balya D, Awatramani GB, Münch TA, Kim DS, Busskamp V, Cepko CL, Roska B. Nat Neurosci. 2008 Jun;11(6):667-75.

11. Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Bi A, Cui J, Ma YP, Olshevskaya E, Pu M, Dizhoor AM, Pan ZH. Neuron. 2006 Apr 6;50(1):23-33.

12. Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness. Doroudchi MM, Greenberg KP, Liu J, Silka KA, Boyden ES, Lockridge JA, Arman AC, Janani R, Boye SE, Boye SL, Gordon GM, Matteo BC, Sampath AP, Hauswirth WW, Horsager A. Mol Ther. 2011 Jul;19(7):1220-9.

13. Influence of blue light on photoreceptors in a live retinal explant system. Roehlecke C, Schumann U, Ader M, Knels L, Funk RH. Mol Vis. 2011 Apr 8;17:876-84.

14. Zhu P, Narita Y, Bundschuh ST, Fajardo O, Schärer YP, Chattopadhyaya B, Bouldoires EA, Stepien AE, Deisseroth K, Arber S, Sprengel R, Rijli FM, Friedrich RW Front Neural Circuits. 2009; 3():21.

15. Gradinaru V, Zhang F, Ramakrishnan C, Mattis J, Prakash R, Diester I, Goshen I, Thompson KR, Deisseroth K Cell. 2010 Apr 2; 141(1):154-165.

16. Grajales-Esquivel E, Luz-Madrigal A, Bierly J, et al. Complement component C3aR constitutes a novel regulator for chick eye morphogenesis. Dev Biol. 2017;428(1):88–100.

17. Ultra light-sensitive and fast neuronal activation with the Ca²+-permeable channelrhodopsin CatCh. Kleinlogel S, Feldbauer K, Dempski RE, Fotis H, Wood PG, Bamann C, Bamberg E. J Biol Chem. 2005 Jun 3;280(22):21061-6.

18. Addendum: independent optical excitation of distinct neural populations.Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK, Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, Constantine-Paton M, Wong GK, Edward S Boyden ES. Nat Methods. 2014 Sep;11(9):972.

Director/a: Eduardo Fernández Jover

codirector/a:Lawrence Humphreys

Publicaciones derivadas de la tesis:
Assessment of Different Niosome Formulations for Optogenetic Applications: Morphological and Electrophysiological Effects, Pharmaceutics (https://doi.org/10.3390/pharmaceutics15071860)

Código ORCID: https://orcid.org/0000-0001-5058-9755