Development of new gene therapy approaches for the treatment of ocular neovascularization

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Cationic polyene phospholipids as DNA carriers for ocular gene therapy

Publication . Machado, Susana; Calado, Sofia; Bitoque, Diogo; Oliveira, Ana Vanessa; Øpstad, Christer L.; Zeeshan, Muhammad; Sliwka, Hans-Richard; Partali, Vassilia; Pungente, Michael D.; Silva, Gabriela

Recent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approach. The eye is a good target due to its small size, minimal diffusion of therapeutic agent to the systemic circulation, and low immune and inflammatory responses. Currently, most approaches are based on viral vectors, but efforts continue towards the synthesis and evaluation of new nonviral carriers to improve nucleic acid delivery. Our objective is to evaluate the efficiency of novel cationic retinoic and carotenoic glycol phospholipids, designated C20-18, C20-20, and C30-20, to deliver DNA to human retinal pigmented epithelium (RPE) cells. Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compounds and coformulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol). Addition of DNA to the liposomes formed lipoplexes, which were characterized for binding, size, biocompatibility, and transgene efficiency. Lipoplex formulations of suitable size and biocompatibility were assayed for DNA delivery, both qualitatively and quantitatively, using RPE cells and a GFP-encoding plasmid. The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable to the known lipid references 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GeneJuice. The results demonstrate that cationic polyene phospholipids have potential as DNA carriers for ocular gene therapy.

2014Journal article

Open access

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2 (Akita)

Publication . Pires De Carvalho, Filipe Ricardo; Calado, Sofia; Silva, Gabriela A.; Diogo, Gabriela S.; Moreira da Silva, Joana; Reis, Rui L.; Cancela, M. Leonor; Gavaia, Paulo

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2 Akita mice that developed T1DM around 3-4 weeks after birth. Both Ins2 Akita and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2 Akita mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2 Akita bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2 Akita . We suggest that Ins2 Akita mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.

2018Journal article

Open access