Patrick T. Yang

In utero gene delivery using chitosan-DNA nanoparticles in mice.

BACKGROUND In utero gene transfer is a novel therapy for monogenic disorders diagnosed in the fetus. Enhanced biosafety alternatives to viral vectors include non-viral transfer agents such as chitosan. The purpose of this study was to evaluate in vitro and in utero gene transfer of reporter gene (GFP) using chitosan as a transfer vehicle. MATERIALS AND METHODS IN VITRO STUDIES 1. Chitosan colloidal suspensions were prepared, and particle stability in murine amniotic fluid (AF) was determined. 2. Chitosan-reporter gene (EGFP) constructs were prepared and protection from endogenous digestion in AF was measured by gel electrophoresis. 3. Transfection efficiency (by chitosan-EGFP) of HEK293T cells was determined in varying proportions of medium and AF. In utero studies: Amniotic sacs of time-mated CD-1 mice were injected with chitosan-pEGFP (12.5 μg DNA) on G17. Pups and their dams were sacrificed and tissues were examined for transgene presence and expression. RESULTS Chitosan formed stable aggregates in AF. Although AF decreased in vitro transfection efficiency, in vivo transfection by amniotic injection achieved short-term transgene expression in pup lung and intestine. CONCLUSIONS In utero delivery of chitosan-EGFP results in postnatal gene expression, and shows promise for non-viral gene transfer in animal models of fetal gene therapy.

Prospective Comparative Analysis of 4 Different Intraocular Pressure Measurement Techniques and Their Effects on Pressure Readings.

Purpose:To compare intraocular pressure (IOP) measurement using the Goldmann applanation tonometry (GAT) without fluorescein, with fluorescein strips, with fluorescein droplets, and IOP measurement with Tono-Pen Avia (TPA). Patients and Methods:This was a prospective comparative clinical analysis. It was performed in clinical practice. The study population consisted of 40 volunteer patients, 1 eye per patient. All patients who were 18 years and older having routine ophthalmological examination were eligible to participate. Active corneal abrasions and/or ulcers, previous glaucoma surgery, or prostheses interfering with GAT measurement were excluded. GAT IOP was measured first without fluorescein, then with fluorescein strip, then with fluorescein droplet, and finally with the TPA device. The main outcome measure was central corneal IOP. Results:Mean±SD IOP measurements for GAT without fluorescein, with fluorescein strip, with fluorescein droplet, and for TPA groups were 12.65±3.01, 14.70±2.82, 15.78±2.64, and 16.33±3.08 mm Hg, respectively. Repeated-measures analysis of variance corrected with the Greenhouse-Geisser estimate ([Latin Small Letter Open E]=0.732) showed that measuring technique had a significant effect on IOP measurements (F2.20,85.59=34.66, P<0.001). The pairwise post hoc testing showed statistically significant mean differences (P⩽0.001) between all techniques except when GAT with fluorescein droplet was compared with TPA (P=0.222). The Bland-Altman analyses showed 95% limits of agreement maximum potential discrepancies in measurement ranging from 5.89 mm Hg in the GAT with fluorescein strip versus droplet compared with 11.83 mm Hg in the GAT with fluorescein strip versus TPA comparison. Conclusions:IOP measurement technique significantly impacted the values obtained. The ophthalmologist should ensure consistent measurement technique to minimize variability when following patients.

Indications for amniotic membrane grafts used in Ontario from 2011 to 2012

Pterygium 46 Neoplasm 9 Persistent epithelial defect 8 Conjunctival surface reconstruction not otherwise specified 5 Chemical burn 5 Glaucoma bleb reconstruction 4 Peripheral corneal melt 3 Vernal or atopic keratoconjunctivitis 2 Corneal melt over lamellar graft 2 Herpes simplex virus corneal epithelial defect 2 Limbal stem cell deficiency 1 Corneal ulcer þ graft versus host disease 1 Fornix reconstruction pemphigoid 1 Graft rejection and corneal thinning after pterygium surgery 1 Keratitis secondary to neurofibromatosis type II 1 Symblepharon 1 Corneal thinning 1 Stevens–Johnson syndrome 1 Strabismus with severe scarring 1 used in Ontario from 2011 to 2012

Descemetorrhexis in endothelial keratoplasty to avoid peripheral bullous keratopathy

Descemet-stripping automated endothelial keratoplasty has revolutionized the treatment of corneal endothelial disease. This procedure requires the removal of the Descemet membrane (DM) and the endothelium from the recipient cornea. We describe a simple technique to perform descemetorrhexis using an ophthalmic viscosurgical device to fill the anterior chamber and using capsulorrhexis forceps to create a controlled, continuous curvilinear DM tear in 19 cases. Avoidance of tearing the DM out to the periphery reduces endothelial cell loss, decreases migration of central endothelium to deficient areas, and minimizes the risk for chronic corneal edema and peripheral bullous keratopathy.

12 DISTRIBUTION AND EXPRESSION OF TRANSGENE GREEN FLUORESCENT PROTEIN IN MICE SURVIVING UP TO 4 WEEKS FOLLOWING IN UTERO GENE THERAPY.

Gene replacement offers a potential cure for degenerative disorders caused by a single gene deletion or mutation. Diagnoses of monogenic disorders in the fetus enable prenatal gene replacement, which may be beneficial from the perspectives of host inflammatory/immune response, efficacy, and disease prevention. Purpose The purpose of our study was to evaluate the distribution and expression of reporter gene green fluorescent protein (GFP) in the tissues of mice surviving up to 1 month following in utero gene therapy. Methods Used Vesicular stomatitis virus-G (VSV-G) pseudotyped lentiviral (LV) vector containing GFP was prepared via triple plasmid cotransfection. Time-mated CD-1 mice underwent individual amniotic sac injection with either 1 × 106 LV particles or saline (controls) on gestational day 16 (term = 21 days) and were allowed to undergo spontaneous parturition. Pups were sacrificed on postnatal days 0, 7, 21, and 28, and neonatal and maternal tissues were analyzed for GFP transgene (by DNA polymerase chain reaction; PCR) and transgene expression by quantitative reverse transcriptase (QRT) PCR and immunohistochemistry (IHC). Summary of Results We observed selective transduction of neonatal tissues (trachea, lung, liver, heart, kidney, spleen, intestine, skeletal muscle) in pups undergoing in utero transfection with LV-GFP. Maternal tissues did not contain transgene despite exposure during amniotic injection. Although the number of pups analyzed at each postnatal time point was small, we observed variable persistence of GFP expression that appeared to be tissue specific (with persistent expression noted in the intestine of 4-week-old pups). Conclusions Neonatal tissue transfection occurs in a variety of tissues following amniotic injection with LV-GFP in this murine model of in utero gene therapy. Transgene persistence and expression patterns observed over the first 4 weeks of life may reflect tissue-specific genomic insertion of transgene that favors persistent transcription in select tissues.

Distribution and expression of transgene green fluorescent protein in mice survived up to four weeks following in utero gene therapys

Gene replacement offers a potential cure for degenerative disorders caused by a single gene deletion or mutation. Diagnoses of monogenic disorders in the fetus enable prenatal gene replacement which may be beneficial from the perspectives of host inflammatory/immune response, efficacy and disease prevention. Purpose: To evaluate the distribution and expression of reporter gene green fluorescent protein (GFP) in the tissues of mice survived up to one month following in utero gene therapy. Methods: Vesicular Stomatitis Virus-G (VSV-G) pseudotyped lentiviral (LV) vector containing GFP was prepared via triple plasmid co-transfection. Time-mated CD-1 mice underwent individual amniotic sac injection with either 1x106 LV particles or saline (controls) on gestational day 16 (term=21d), and were allowed to undergo spontaneous parturition. Pups were sacrificed on postnatal days 0, 7, 21 and 28, and neonatal and maternal tissues were analyzed for GFP transgene (by DNA polymerase chain reaction; PCR), and transgene expression by quantitative reverse transcriptase (QRT) PCR and immunohistochemistry (IHC). Results: We observed selective transduction of neonatal tissues (trachea, lung, liver, heart, kidney, spleen, intestine, skeletal muscle), in pups undergoing in utero transfection with LV-GFP. Maternal tissues did not contain transgene despite exposure during amniotic injection. Although the numbers of pups analyzed at each postnatal time point was small, we observed variable persistence of GFP expression that appeared to be tissue specific (with persistent expression noted in intestine of 4 week old pups). Conclusions: Neonatal tissue transfection occurs in a variety of tissues following amniotic injection with LV-GFP in this murine model of in utero gene therapy. Transgene persistence and expression patterns observed over the first 4 weeks of life may reflect tissue-specific genomic insertion of transgene that favors persistent transcription in select tissues.