Development and preclinical evaluation of a novel AAV vector-based gene therapy to treat retinitis pigmentosa type 45

Wagner, Johanna (2021): Development and preclinical evaluation of a novel AAV vector-based gene therapy to treat retinitis pigmentosa type 45. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)

Abstract

Loss-of-function mutations in the cyclic nucleotide-gated channel beta 1 subunit (Cngb1) gene are known to cause Retinitis pigmentosa type 45 (RP45), an incurable retinal disorder characterized by primary functional loss and degeneration of rod photoreceptors, followed by a non-cell autonomous cone death, often resulting in legal blindness. Here, a novel recombinant adeno-associated virus vector for gene supplementation therapy of Cngb1-linked RP was developed and tested for its efficacy in two preclinical Cngb1 knockout (KO) animal models of RP45. The full-length human CNGB1 coding sequence was packaged in recombinant AAV5 under the control of a short human rhodopsin promoter, as CNGB1 is expressed natively in rod photoreceptors. Due to the limited cargo capacity of the AAV, an optimized shortened rhodopsin promoter was designed (hRHO194) in order to produce the entire expression cassette in cis and to ensure rod-specific expression of hCNGB1. The resulting viral vector (rAAV5.hCNGB1) was produced by triple-transfection in HEK293 cells as well as in HeLa cells as a producer cell line. For the preclinical validation of the therapy, the efficacy of rAAV5.hCNGB1 was assessed in a Cngb1 KO mouse model as well as in a Cngb1 KO dog model. The animals were treated with rAAV5.CNGB1 delivered via subretinal injection to assess transgene expression as well as biological activity. The effect of the treatment was read out with multiple outcome measures. Proper availability, integrity, and localization of the introduced gene product was assessed by immunohistochemistry. Retinal function was examined via ERG measurements and morphological effects on photoreceptor degeneration were monitored in vivo by OCT imaging. Behavioral tests were implemented to validate visual function. Additionally, critical aspects including studies on long-term efficacy, biologically active dose range, time window for a therapeutic intervention, as well as on potential toxicities were addressed, to provide further insights on the feasibility and safety of the drug administration. The study demonstrated efficient, specific, and long-term hCNGB1 expression in murine and canine rod photoreceptors driven by the short hRHO194 promoter. By treatment with rAAV5.hCNGB1, a substantial preservation of rod and cone photoreceptors was achieved in both Cngb1 KO mice and dogs. Furthermore, the efficacy of rAAV5.hCNGB1 was dose-dependent manifesting itself as an increasing expression level with ascending dose, resulting in enhanced biological efficacy in both animal models. In addition, a toxic side effect was identified for the vector produced in HeLa cells emerging in a dose-dependent manner, which was not observed for the vector produced in HEK293 cells. It was further shown, that the viral vector-introduced human CNGB1 subunit was capable of forming functional chimeric rod-specific CNG channels together with the endogenous murine or canine CNGA1 subunits leading to a recovery of rod photoreceptor function as well as markedly improved vision-guided behavior in both animal models. Furthermore, retinal stress in the Cngb1 KO mouse, manifesting as Müller cell gliosis, was reduced by rAAV5.hCNGB1, even beyond the treated region of the retina. Finally, it was shown that a degeneration-induced inflammation was diminished by the treatment in the Cngb1 KO dog model, shown as reduced microglia activation, while in one treated dog an increased immune reaction was observed, manifesting as increase activation of microglia within the retina. These results demonstrated the efficacy of the rAAV5.hCNGB1 gene supplementation therapy in small and large animals and thus showed the transferability of this approach to human application. Thereby, the work covered a major part of the preclinical phase and proved that rAAV5.hCNGB1 is a suitable candidate for a clinical application. The results of this study provided novel insights into the transferability of a gene therapy from mouse to man and will serve as a basis for a design of preclinical toxicology studies and early-phase clinical application of rAAV5.hCNGB1.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Gene Therapy, Retinitis Pigmentosa, Recombinant AAV, Vector Construction, Animal Model
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Graduate School of Systemic Neurosciences (GSN)
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 3. März 2021
• 1. Bericht­erstatter:in: Michalakis, Stylianos