Drug Delivery to Posterior Intraocular Tissues: Third Annual ARVO/Pfizer Ophthalmics Research Institute Conference

The third Annual ARVO/Pfizer Ophthalmic Research Institute Conference was held Friday and Saturday, May 4 and 5, 2007 at the Fort Lauderdale Grande Hotel and Yacht Club, Fort Lauderdale, Florida. The conference, funded by the ARVO Foundation for Eye Research through a grant from Pfizer Ophthalmics, provided an opportunity to gather experts from within and outside ophthalmology to develop strategies to address drug delivery to posterior intraocular tissues—a topic of great interest, as the major route of drug delivery is via intravitreous injection

Voluntary exercise modulates pathways associated with amelioration of retinal degenerative diseases

Background: Exercise has been shown to promote a healthier and longer life and linked to a reduced risk of developing neurodegenerative diseases including retinal degenerations. However, the molecular pathways underpinning exercise-induced cellular protection are not well understood. In this work we aim to profile the molecular changes underlying exercise-induced retinal protection and investigate how exercise-induced inflammatory pathway modulation may slow the progression of retinal degenerations. Methods: Female C57Bl/6J mice at 6 weeks old were given free access to open voluntary running wheels for a period of 28 days and then subjected to 5 days of photo-oxidative damage (PD)-induced retinal degeneration. Following, retinal function (electroretinography; ERG), morphology (optical coherence tomography; OCT) and measures of cell death (TUNEL) and inflammation (IBA1) were analysed and compared to sedentary controls. To decipher global gene expression changes as a result of voluntary exercise, RNA sequencing and pathway and modular gene co-expression analyses were performed on retinal lysates of exercised and sedentary mice that were subjected to PD, as well as healthy dim-reared controls. Results: Following 5 days of PD, exercised mice had significantly preserved retinal function, integrity and reduced levels of retinal cell death and inflammation, compared to sedentary controls. In response to voluntary exercise, inflammatory and extracellular matrix integrity pathways were significantly modulated, with the gene expression profile of exercised mice more closely trending towards that of a healthy dim-reared retina. Conclusion: We suggest that voluntary exercise may mediate retinal protection by influencing key pathways involved in regulating retinal health and shifting the transcriptomic profile to a healthy phenotype

Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina

PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Tauroursodeoxycholic Acid Protects Retinal and Visual Function in a Mouse Model of Type 1 Diabetes

Purpose: Previous studies demonstrated that systemic treatment with tauroursodeoxycholic acid (TUDCA) is protective in in vivo mouse models of retinal degeneration and in culture models of hyperglycemia. This study tested the hypothesis that TUDCA will preserve visual and retinal function in a mouse model of early diabetic retinopathy (DR). Methods: Adult C57BL/6J mice were treated with streptozotocin (STZ) and made diabetic at 8–10 weeks of age. Control and diabetic mice were treated with vehicle or TUDCA starting 1 or 3 weeks after induction of diabetes, and were assessed bimonthly for visual function via an optomotor response and monthly for retinal function via scotopic electroretinograms. Results: Diabetic mice showed significantly reduced spatial frequency and contrast sensitivity thresholds compared to control mice, while diabetic mice treated early with TUDCA showed preservation at all timepoints. A-wave, b-wave, and oscillatory potential 2 (OP2) amplitudes decreased in diabetic mice. Diabetic mice also exhibited delays in a-wave and OP2-implicit times. Early TUDCA treatment ameliorated a-wave, b-wave, and OP2 deficits. Late TUDCA treatment showed reduced preservation effects compared to early treatment. Conclusions: Early TUDCA treatment preserved visual function in an STZ-mouse model of Type I diabetes. These data add to a growing body of preclinical research that may support testing whether TUDCA may be an effective early clinical intervention against declining visual function caused by diabetic retinopathy