This proposal seeks to develop triplex oligonucleotide (known as """"""""triplex oligo"""""""", """"""""TFO"""""""", or """"""""antigene"""""""") technology into a useful therapy for herpes keratitis. Triplex oligo is based on the observation that a third DNA strand - the triplex - can bind non-covalently to specific double-stranded DNA target sequences to induce mutation or affect transcription. We have identified several such target sequences within the HSV-1 genome, including two in the latency-associated transcript (LAT) region. Targeting LAT, a crucial part of the HSV genome, is proposed as a logical first step toward the prevention of HSV-1 gene transcription and, hence, ocular reactivation. This novel genetic therapy could potentially prevent thousands of episodes of recurrent herpes keratitis each year. A large amount of preliminary data is presented to support the feasibility of the proposed studies.
The specific aims of the proposal are: 1. Mechanism of action of photoactivatable TFO's. Control pheophorbide conjugated TFO's matched to TFO-1 and TFO-3 in length and AG composition will be sythesized. TFO-1-pheo and TFO-3-pheo alone and in combination will be retested using the appropriate control oligonucleotides. The mechanism of antiviral activity of the pheophorbide-linked TFO's will be investigated by specific cleavage site determinations, guanine oxidation, and cross-linking experiments. 2. Delivery of anti-HSV-1 Triplex-Forming Oligonucleotides. Various strategies for delivery of anti-HSV-1 TFO's into the TG's of live mice will be examined with an emphasis on intraperitoneal and/or intravenous infusion of oligonucleotides. The integrity of the delivered oligos will be determined by native gel electrophoresis of TG cellular extracts. 3. Prevention of HSV-1 Ocular Reactivation. The mouse model of HSV-1 ocular infection and reactivation will be used to investigate whether triplex oligos can inhibit HSV-1 induced ocular reactivation. An ex vivo model of HSV-1 reactivation in explanted mouse trigeminal ganglia is proposed as an efficient means for determining effective strategies for the delay or inhibition of reactivation.
