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Development of gene therapy for treatment of age-related macular degeneration.
Acta Ophthalmol 2014; 92 Thesis3:1-38AO

Abstract

Intraocular neovascular diseases are the leading cause of blindness in the Western world in individuals over the age of 50. Age-related macular degeneration (AMD) is one of these diseases. Exudative AMD, the late-stage form, is characterized by abnormal neovessel development, sprouting from the choroid into the avascular subretinal space, where it can suddenly cause irreversible damage to the vulnerable photoreceptor (PR) cells essential for our high-resolution, central vision. The molecular basis of AMD is not well understood, but several growth factors have been implicated including vascular endothelial growth factor (VEGF), and the advent of anti-VEGF therapy has markedly changed the outcome of treatment. However, common to all current therapies for exudative AMD are the complications of repeated monthly intravitreal injections, which must be continued throughout one's lifetime to maintain visual benefits. Additionally, some patients do not benefit from established treatments. Strategies providing long-term suppression of inappropriate ocular angiogenesis are therefore needed, and gene therapy offers a potential powerful technique. This study aimed to develop a strategy based on RNA interference (RNAi) for the sustained attenuation of VEGF. We designed a panel of anti-VEGF short hairpin RNAs (shRNA), and based on the most potent shRNAs, microRNA (miRNA)-mimicked hairpins were developed. We demonstrated an additive VEGF silencing effect when we combined the miRNAs in a tricistronic miRNA cluster. To meet the requirements for development of medical treatments for AMD with long-term effects, the shRNA/miRNA is expressed from vectors based on adeno-associated virus (AAV) or lentivirus (LV). Both vector systems have been found superior in terms of transduction efficiency and persistence in gene expression in retinal cells. The capacity of AAV-encoded RNAi effector molecules to silence endogenous VEGF gene expression was evaluated in mouse models, including the model of laser-induced choroidal neovascularization (CNV), and we found that subretinal administration of self-complementary (sc)-AAV2/8 encoding anti-VEGF shRNAs can impair vessel formation. In parallel, a significant reduction of endogenous VEGF was demonstrated following injection of scAAV2/8 vectors expressing multiple anti-VEGF miRNAs into murine hind limb muscles. Furthermore, in an ongoing project we have designed versatile, multigenic LV vectors with combined expression of multiple miRNAs and proteins, including pigment epithelium-derived factor (PEDF), a multifunctional, secreted protein that has anti-angiogenic and neurotrophic functions. Co-expression of miRNAs and proteins from a single viral vector increases safety by minimizing the viral load necessary to obtain a therapeutic effect and thereby reduces the risk of insertional mutagenesis as well as the immune response against viral proteins. Our results show co-expression of functional anti-VEGF-miRNAs and PEDF in cell studies, and in vivo studies reveal an efficient retinal pigment epithelium (RPE)-specific gene expression following the incorporation of the vitelliform macular dystrophy 2 (VMD2) promoter, demonstrating the potential applicability of our multigenic LV vectors in ocular anti-VEGF gene therapy, including combination therapy for treatment of exudative AMD. In conclusion, these highly promising data clearly demonstrate that viral-encoded RNAi effector molecules can be used for the inhibition of neovascularization and will, in combination with the growing interest of applying DNA- or RNA-based technologies in the clinic, undoubtedly contribute to the development of efficacious long-term gene therapy treatment of intraocular neovascular diseases.

Authors+Show Affiliations

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

24953666

Citation

Askou, Anne Louise. "Development of Gene Therapy for Treatment of Age-related Macular Degeneration." Acta Ophthalmologica, vol. 92 Thesis3, 2014, pp. 1-38.
Askou AL. Development of gene therapy for treatment of age-related macular degeneration. Acta Ophthalmol. 2014;92 Thesis3:1-38.
Askou, A. L. (2014). Development of gene therapy for treatment of age-related macular degeneration. Acta Ophthalmologica, 92 Thesis3, pp. 1-38. doi:10.1111/aos.12452.
Askou AL. Development of Gene Therapy for Treatment of Age-related Macular Degeneration. Acta Ophthalmol. 2014;92 Thesis3:1-38. PubMed PMID: 24953666.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of gene therapy for treatment of age-related macular degeneration. A1 - Askou,Anne Louise, PY - 2014/6/24/entrez PY - 2014/6/24/pubmed PY - 2015/2/13/medline SP - 1 EP - 38 JF - Acta ophthalmologica JO - Acta Ophthalmol VL - 92 Thesis3 N2 - Intraocular neovascular diseases are the leading cause of blindness in the Western world in individuals over the age of 50. Age-related macular degeneration (AMD) is one of these diseases. Exudative AMD, the late-stage form, is characterized by abnormal neovessel development, sprouting from the choroid into the avascular subretinal space, where it can suddenly cause irreversible damage to the vulnerable photoreceptor (PR) cells essential for our high-resolution, central vision. The molecular basis of AMD is not well understood, but several growth factors have been implicated including vascular endothelial growth factor (VEGF), and the advent of anti-VEGF therapy has markedly changed the outcome of treatment. However, common to all current therapies for exudative AMD are the complications of repeated monthly intravitreal injections, which must be continued throughout one's lifetime to maintain visual benefits. Additionally, some patients do not benefit from established treatments. Strategies providing long-term suppression of inappropriate ocular angiogenesis are therefore needed, and gene therapy offers a potential powerful technique. This study aimed to develop a strategy based on RNA interference (RNAi) for the sustained attenuation of VEGF. We designed a panel of anti-VEGF short hairpin RNAs (shRNA), and based on the most potent shRNAs, microRNA (miRNA)-mimicked hairpins were developed. We demonstrated an additive VEGF silencing effect when we combined the miRNAs in a tricistronic miRNA cluster. To meet the requirements for development of medical treatments for AMD with long-term effects, the shRNA/miRNA is expressed from vectors based on adeno-associated virus (AAV) or lentivirus (LV). Both vector systems have been found superior in terms of transduction efficiency and persistence in gene expression in retinal cells. The capacity of AAV-encoded RNAi effector molecules to silence endogenous VEGF gene expression was evaluated in mouse models, including the model of laser-induced choroidal neovascularization (CNV), and we found that subretinal administration of self-complementary (sc)-AAV2/8 encoding anti-VEGF shRNAs can impair vessel formation. In parallel, a significant reduction of endogenous VEGF was demonstrated following injection of scAAV2/8 vectors expressing multiple anti-VEGF miRNAs into murine hind limb muscles. Furthermore, in an ongoing project we have designed versatile, multigenic LV vectors with combined expression of multiple miRNAs and proteins, including pigment epithelium-derived factor (PEDF), a multifunctional, secreted protein that has anti-angiogenic and neurotrophic functions. Co-expression of miRNAs and proteins from a single viral vector increases safety by minimizing the viral load necessary to obtain a therapeutic effect and thereby reduces the risk of insertional mutagenesis as well as the immune response against viral proteins. Our results show co-expression of functional anti-VEGF-miRNAs and PEDF in cell studies, and in vivo studies reveal an efficient retinal pigment epithelium (RPE)-specific gene expression following the incorporation of the vitelliform macular dystrophy 2 (VMD2) promoter, demonstrating the potential applicability of our multigenic LV vectors in ocular anti-VEGF gene therapy, including combination therapy for treatment of exudative AMD. In conclusion, these highly promising data clearly demonstrate that viral-encoded RNAi effector molecules can be used for the inhibition of neovascularization and will, in combination with the growing interest of applying DNA- or RNA-based technologies in the clinic, undoubtedly contribute to the development of efficacious long-term gene therapy treatment of intraocular neovascular diseases. SN - 1755-3768 UR - https://www.unboundmedicine.com/medline/citation/24953666/Development_of_gene_therapy_for_treatment_of_age_related_macular_degeneration_ L2 - https://doi.org/10.1111/aos.12452 DB - PRIME DP - Unbound Medicine ER -