Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

Cytosolic StAR-related lipid transfer domain 4 (STARD4) protein influences keratinocyte lipid phenotype and differentiation status.

BACKGROUND: Terminally differentiating keratinocytes actively synthesize and accumulate cholesterol, which is a key constituent of intercellular lipid lamellae which contribute to the epidermal permeability barrier. While the pathway for cholesterol biosynthesis is established, intracellular transport mechanisms for this lipid are poorly understood, despite their importance in regulating organelle sterol content, keratinocyte differentiation status and the activity of lipid-responsive transcription factors involved in skin health, repair and disease. Recent data implicate proteins containing a steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain in cellular cholesterol homeostasis. OBJECTIVES: To investigate gene expression of cytosolic, cholesterol-binding StAR-related lipid transfer domain 4 (STARD4) protein in primary human keratinocytes and differentiating HaCaT keratinocytes and, by overexpression of this protein, the function of STARD4 in HaCaT keratinocyte lipid phenotype and differentiation status. METHODS: Quantitative polymerase chain reaction was utilized to measure gene expression of STARD4 relative to the housekeeping gene GAPDH. Following transient (48 h) overexpression of STARD4, keratinocyte lipid mass and lipogenesis were measured, along with expression of genes involved in cholesterol homeostasis and those encoding a range of keratinocyte differentiation markers. RESULTS: Cholesterol-binding protein STARD4 is expressed in both primary and immortalized HaCaT keratinocytes, and is repressed during Ca(2+) -dependent differentiation of the latter. Transient overexpression of STARD4 reduces endogenous [(14) C]cholesterol and cholesteryl ester biosynthesis, and triggers increased expression of SREBF2, ABCG4 and LOR, while repressing expression of ABCA1. CONCLUSIONS: The cytosolic cholesterol-sensing protein STARD4 modulates both keratinocyte cholesterol homeostasis and differentiation status, increasing the efficiency of cholesterol trafficking within the cell, and amplifying and 'fine-tuning' cellular responses to this sterol. Modulation of expression of STARD4, and other members of the START family of lipid trafficking proteins, may prove useful in resolving imbalances in lipid metabolism associated with loss of epidermal barrier function in psoriasis and atopic dermatitis.