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Retinal gene therapy using adeno-associated viruses (AAVs) is constrained by the mode of viral vector delivery. Intravitreal AAV injections are impeded by the internal limiting membrane barrier, while subretinal injections require invasive surgery and produce a limited region of therapeutic effect. In this study, we introduce a novel mode of ocular gene delivery in rhesus macaques using transscleral microneedles to inject AAV8 into the subretinal or suprachoroidal space, a potential space between the choroid and scleral wall of the eye. Using in vivo imaging, we found that suprachoroidal AAV8 produces diffuse, peripheral expression in retinal pigment epithelial (RPE) cells, but it elicited local infiltration of inflammatory cells. Transscleral subretinal injection of AAV8 using microneedles leads to focal gene expression with transduction of RPE and photoreceptors, and minimal intraocular inflammation. In comparison, intravitreal AAV8 shows minimal transduction of retinal cells, but elicits greater systemic humoral immune responses. Our study introduces a novel mode of transscleral viral delivery that can be performed without vitreoretinal surgery, with focal or diffuse transgene expression patterns suitable for different applications. The decoupling of local and systemic immune responses reveals important insights into the immunological consequences of AAV delivery to different ocular compartments surrounding the blood-retinal barrier.

Purpose: To evaluate choroidal and suprachoroidal changes following suprachoroidal injection of triamcinolone acetonide injectable suspension (CLS-TA), in eyes with macular edema due to retinal vein occlusion (RVO). Design: Prospective cohort study within a randomized, controlled phase 2 clinical trial. Methods: Enhanced depth imaging optical coherence tomography (EDI-OCT) images were analyzed from 38 eyes of 38 treatment-naïve patients with macular edema due to RVO, enrolled in the prospective Suprachoroidal Injection of Triamcinolone Acetonide with Intravitreal Aflibercept in Subjects with Macular Edema Due to Retinal Vein Occlusion (TANZANITE) study who received either a suprachoroidal injection of CLS-TA with an intravitreal injection of aflibercept (combination arm) or only an intravitreal injection of aflibercept (monotherapy arm), followed by monthly intravitreal aflibercept injections in both arms based on pro re nata criteria. Results: Macular choroidal thickness measured to the outer choroidal vessel lumen (vascular choroidal thickness, VCT), outer choroid stroma (stromal choroidal thickness, SCT), or inner scleral border (total choroidal thickness, TCT) showed no significant changes over 3 months in both study arms (P =.231-.342). Eyes that received combination therapy showed a trend toward thickening of the suprachoroidal space (SCS) compared with monotherapy alone (13.4 μm vs 5.3 μm at 3 months; P =.077). In the 15 eyes that demonstrated a visible SCS at baseline, the SCS expanded significantly after suprachoroidal CLS-TA injection (16.2 μm to 27.8 μm at 3 months; P =.033). Conclusions: Suprachoroidal injection of CLS-TA does not alter choroidal thickness in eyes with macular edema due to RVO, but may result in expansion of the SCS.

Retinal and choroidal vascular diseases, with their associated abnormalities in vascular permeability, account for the majority of patients with vision loss in industrialized nations. VEGF is upregulated in ischemic retinopathies such as diabetes and is known to dramatically alter vascular permeability in a number of nonocular tissues via Src kinase--regulated signaling pathways. VEGF antagonists are currently in clinical use for treating the new blood vessels and retinal edema associated with neovascular eye diseases, but such therapies require repeated intraocular injections. We have found that vascular leakage following intravitreal administration of VEGF in mice was abolished by systemic or topical delivery of what we believe is a novel VEGFR2/Src kinase inhibitor; this was confirmed in rabbits. The relevance of Src inhibition to VEGF-associated alterations in vascular permeability was further substantiated by genetic studies in which VEGF injection or laser-induced vascular permeability failed to augment retinal vascular permeability in Src-/- and Yes-/- mice (Src and Yes are ubiquitously expressed Src kinase family members; Src-/- and Yes-/- mice lacking expression of these kinases show no vascular leak in response to VEGF). These findings establish a role for Src kinase in VEGF-mediated retinal vascular permeability and establish a potentially safe and painless topically applied therapeutic option for treating vision loss due to neovascular-associated retinal edema., Introduction Abnormal retinal vascular permeability leading to edema in the area of the macula is the leading cause of vision loss in diseases such as diabetic retinopathy, exudative macular degeneration, [...]

Although phosphoinositide 3-kinases (PI3Ks) play beneficial pro-cell survival roles during tissue ischemia, some isoforms ([gamma], and [delta]) paradoxically contribute to the inflammation that damages these same tissues upon reperfusion. We therefore considered the possibility that selectively inhibiting proinflammatory PI3K isoforms during the reperfusion phase could ultimately limit overall tissue damage seen in ischemia/reperfusion injuries such as myocardial infarction. Panreactive and isoform-restricted PI3K inhibitors were identified by screening a novel chemical family; molecular modeling studies attributed isoform specificity based on rotational freedom of substituent groups. One compound (TG100-115) identified as a selective PI3K [gamma]/3 inhibitor potently inhibited edema and inflammation in response to multiple mediators known to participate in myocardial infarction, including vascular endothelial growth factor and platelet-activating factor; by contrast, endothelial cell mitogenesis, a repair process important to tissue survival after ischemic damage, was not disrupted. In rigorous animal MI models, TG100-115 provided potent cardioprotection, reducing infarct development and preserving myocardial function. Importantly, this was achieved when dosing well after myocardial reperfusion (up to 3 h after), the same time period when patients are most accessible for therapeutic intervention. In conclusion, by targeting pathologic events occurring relatively late in myocardial damage, we have identified a potential means of addressing an elusive clinical goal: meaningful cardioprotection in the postreperfusion time period. edema | inflammation | myocardial infarct | VEGF

A hydrogen bonded dimer having an unprecedented 16-membered ring is identified during the study of several different types of (2-cyanoethyl)urea derivatives. The process of hydrogen bonding is essential for the production of structured assemblies of molecules. Hydrogen bonded-dimes are always made up of minimum of cyclic eight arrays of atoms.

A better procedure in the generation of enantiomerically pure amino acid ester isocyanates is presented. The new method no longer calls for the use of gaseous phosgene, high temperature conditions and very high hydronium ion concentrations. A solution of phosgene in toluene replaces gaseous toluene and removes the hydrogen chloride in solution. The isocyanate derived from amino acid ester hydrochloride and pyridine precursors is purified by aqueous extraction at freezing point followed by Kugelrohr distillation.

Intramolecular hydrogen bonding plays a key role in the conformation of oligoureas. The urea derivatives are obtained by an iterative process that involves the conjugate addition of a primary amine to acrylonitrile, followed by reaction with an isocyanate then by reduction of the nitrile group to form a primary amine. Spectroscopic studies reveal significant intramolecular hydrogen bonding of the NH group with the carbonyl oxygen. Moreover, N-alkyl and aryl substituents exert a strong influence on the orientation of the carbonyl groups. This conformational control makes oligoureas potentially useful as molecular scaffoldings or oligoamine backbones.

The synthesis of a two-stranded parallel beta-sheet, in which the dipeptide strands are attached to a a diurea molecular scaffold, is described. The synthesis involves the sequential treatment of diamine with peptide isocyanates, followed by aminolysis of the methyl ester groups with methylamine. Nuclear Overhauser effect and chemical shift studies showed that the artificial beta-sheets adopt a parallel conformation in chloroform solution.

Retinal gene therapy using adeno-associated viruses (AAVs) is constrained by the mode of viral vector delivery. Intravitreal AAV injections are impeded by the internal limiting membrane barrier, while subretinal injections require invasive surgery and produce a limited region of therapeutic effect. In this study, we introduce a novel mode of ocular gene delivery in rhesus macaques using transscleral microneedles to inject AAV8 into the subretinal or suprachoroidal space, a potential space between the choroid and scleral wall of the eye. Using in vivo imaging, we found that suprachoroidal AAV8 produces diffuse, peripheral expression in retinal pigment epithelial (RPE) cells, but it elicited local infiltration of inflammatory cells. Transscleral subretinal injection of AAV8 using microneedles leads to focal gene expression with transduction of RPE and photoreceptors, and minimal intraocular inflammation. In comparison, intravitreal AAV8 shows minimal transduction of retinal cells, but elicits greater systemic humoral immune responses. Our study introduces a novel mode of transscleral viral delivery that can be performed without vitreoretinal surgery, with focal or diffuse transgene expression patterns suitable for different applications. The decoupling of local and systemic immune responses reveals important insights into the immunological consequences of AAV delivery to different ocular compartments surrounding the blood-retinal barrier., (© 2020 The Author(s).)