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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Small Research Grants (R03)
Project #
5R03EY013799-03
Application #
7123811
Study Section
Special Emphasis Panel (ZEY1-VSN (01))
Program Officer
Shen, Grace L
Project Start
2004-08-01
Project End
2008-07-31
Budget Start
2006-08-01
Budget End
2008-07-31
Support Year
3
Fiscal Year
2006
Total Cost
$145,987
Indirect Cost
Name
University of Utah
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112