Targeting the Nrf2 Signaling Pathway in the Retina With a Gene-Delivered Secretable and Cell-Penetrating Peptide.

Abstract

PURPOSE:

Oxidative stress has been linked to several ocular diseases, initiating an inflammatory response that increases tissue injury. The Nrf2 transcription factor regulates expression of antioxidant genes and is tightly regulated by Kelch-Like ECH-Associated Protein 1 (Keap-1). We evaluate the antioxidant and anti-inflammatory properties of an adeno-associated virus (AAV) vector delivering an Nrf2-derived peptide that binds Keap-1.

METHODS:

The sequence of the Nrf2 peptide was fused to a cell-penetrating peptide (Tat-peptide) sequence (TatNrf2mer). The effects of lentiviral-delivered TatNrf2mer were studied in vitro. Transcript (quantitative [q] RT-PCR) and protein levels (ELISA and immunofluorescence) were quantified. Cell viability was measured by MTT and Cell Titer assays. The AAV vectors were packaged with the TatNrf2mer fused to secretable green fluorescent protein (GFP) under the control of the small chicken β actin promoter. The protective effects of this vector were evaluated in a model of RPE oxidative injury and in a mouse model of uveitis after intravitreal injection.

RESULTS:

Expression of TatNrf2mer peptide induced antioxidant gene expression, blocked IL-1β secretion, and protected cells from oxidative injury. In mice, TatNrf2mer expression partially protected photoreceptor function based on ERG responses and optical coherence tomography measurements in the sodium iodate (NaIO3) model. Furthermore, sGFP-TatNrf2mer expression decreased IL-1β and IL-6 in the NaIO3-treated mice, and resulted in a 54% decrease in the number of inflammatory cells in the vitreous body of the endotoxin-induced uveitis mouse model.

CONCLUSIONS:

The intravitreally delivered AAV-TatNrf2mer has antioxidant and anti-inflammatory effects in widely-used models of ocular injury, suggesting it also could be useful in ocular diseases associated with oxidative stress and inflammasome activation.

• Authors: Cristhian J Ildefonso, Henrique Jaime, Emily E Brown, Ryo L Iwata, Chulbul M Ahmed, Michael T Massengill, Manas R Biswal, Shannon E Boye, William W Hauswirth, John D Ash, Qiuhong Li, Alfred S Lewin
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 57 Issue 2 Pg. 372-86 (Feb 2016) ISSN: 1552-5783 [Electronic] United States
• PMID: 26842755 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Cell-Penetrating Peptides
• Interleukin-1beta
• Intracellular Signaling Peptides and Proteins
• KEAP1 protein, human
• Kelch-Like ECH-Associated Protein 1
• NF-E2-Related Factor 2
• NFE2L2 protein, human
• Green Fluorescent Proteins

Topics

• Animals
• Cell Line
• Cell-Penetrating Peptides (genetics)
• Dependovirus (genetics)
• Disease Models, Animal
• Enzyme-Linked Immunosorbent Assay
• Fluorescent Antibody Technique, Indirect
• Genetic Therapy
• Genetic Vectors
• Geographic Atrophy
• Green Fluorescent Proteins (genetics)
• Humans
• Interleukin-1beta (metabolism)
• Intracellular Signaling Peptides and Proteins (genetics)
• Kelch-Like ECH-Associated Protein 1
• Male
• Mice
• Mice, Inbred C57BL
• NF-E2-Related Factor 2 (genetics)
• Oxidative Stress
• Real-Time Polymerase Chain Reaction
• Retina (metabolism)
• Retinal Pigment Epithelium (metabolism)
• Signal Transduction
• Transfection
• Transgenes
• Uveitis