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Table of contents
INTRODUCTION
I. RETINITIS PIGMENTOSA INDUCED BY RHODOPSIN MUTATION
I.1. EYE AND VISION
I.1.a. Generalities
I.1.b. The retina
I.1.c. Human photoreceptors
I.2. RETINITIS PIGMENTOSA
I.2.a. Remodeling of the retina and clinical manifestations
I.2.b. Genetic involvement
I.3. RHODOPSIN
I.3.a. Rhodopsin synthesis, function and visual cycle
I.3.b. Structure of rhodopsin
I.3.c. Rhodopsin mutations and consequences
I.4. TREATMENTS OF RP
I.4.a. General current treatments of RP
I.4.b. Treatments of RP in the particular case of RHO mutation
II. CURRENT STRATEGIES TO PREVENT MUTANT RHODOPSIN EXPRESSION
II.1. RHODOPSIN DNA SEQUENCE CORRECTION
II.1.a. DNA repair by Homologous Recombination (HR)
II.1.b. HR-induced endonucleases
II.2. SILENCING OF MUTANT GENE EXPRESSION COUPLED TO EXPRESSION OF A NORMAL COPY
II.2.a. Zinc Finger – Artificial Transcription Factors (ZF-ATF)
II.2.b. RNA interference
II.2.c. Ribozymes
III. TRANS-SPLICING OF RHODOPSIN PRE-MRNA
III.1. CIS-SPLICING: THE PROTOTYPICAL SPLICE REACTION
III.1.a. Splicing is a phase of pre-mRNA maturation
III.1.b. Steps of the splicing mechanism
III.1.c. Regulation of cis-splicing
III.2. TRANS-SPLICING BETWEEN TWO RNA MOLECULES
III.2.a. Description of the mechanism
III.2.b. Previous studies led with trans-splicing approaches
III.2.c. Strategies of PTM Design
III.2.d. Advantages and drawbacks
IV. VIRAL VECTORS FOR GENE THERAPY OF OCULAR DISEASES
IV.1. ADVANTAGES OF THE EYE AS TARGET OF GENE THERAPY WITH VIRAL VECTOR
IV.2. CHOICE OF THE VIRAL VECTOR FOR OCULAR DISEASES
IV.2.a. Classical viral vectors used in gene therapies
IV.2.b. Previous ocular gene therapies with rAAV
V. CELLULAR AND ANIMAL MODELS OF RP LINKED TO RHODOPSIN MUTATIONS.
V.1. CELLULAR MODEL OF RHODOPSIN EXPRESSION
V.1.a. Primary cultures of photoreceptors
V.1.b. Cell lines developed to study rhodopsin mutations
V.2. ANIMAL MODELS OF RHODOPSIN-INDUCED RETINITIS PIGMENTOSA
V.2.a. Animal models of rhodopsin mutation-induced retinitis pigmentosa and more specifically mouse models
V.2.b. Comparison of mouse and human eyes
RESULTS
I. RESULTS OF IN VITRO ANALYSIS OF PTM EFFICIENCY.
II. IMPROVEMENT AND CHARACTERIZATION OF AN IN VIVO IMAGING TECHNOLOGY TO
MEASURE POTENTIAL EFFECTS OF OUR THERAPEUTIC STRATEGY IN A HUMANIZED MOUSE MODEL.
III. COMPLEMENTARY ONGOING RESULTS.
III.1. DEVELOPMENT OF NEW PTM IN VITRO AND STRATEGIES TO OVERCOME PTM DRAWBACKS.
III.1.a. A new PTM that repairs the fifth exon.
III.1.b. Production of a truncated protein in vitro after expression of the PTM alone.
III.1.c. Strategies to prevent production of truncated protein.
III.1.d. Analysis of PTM translation alone in vivo.
III.2. STUDY OF THE THERAPEUTIC EFFECTS OF TRANS-SPLICING IN A HUMANIZED MOUSE MODEL
III.2.a. The humanized Rho+/- P347S RHO+ mouse model.
III.2.b. Choice of parameters that regulate PTM expression in vivo.
III.2.c. Effect of AAV2/8 bRho-PTM20 injection in Rho+/- P347S RHO+ mice.
DISCUSSION AND PERSPECTIVES
I. THE PTM SEQUENCE: THE KEY TO EFFICIENT SMART TECHNOLOGY.
II. HOW TO ACHIEVE MORE TRANS-SPLICING THAN CIS-SPLICING?
III. THE MAIN DRAWBACKS OF PTM.
IV. FOLLOWING-UP POTENTIAL BENEFICIAL EFFECTS IN A HUMANIZED MOUSE MODEL
V. ENVISAGING A THERAPEUTIC APPLICATION IN HUMANS.
VI. THE FUTURE OF THERAPEUTIC STRATEGIES TO PREVENT MUTANT RHODOPSIN EXPRESSION.
VII. CONSIDERING GENE THERAPIES FOR RETINAL DISEASES OVER THE NEXT FEW DECADES.
BIBLIOGRAPHY
ANNEXES
