23 research outputs found
Corneas of scAAV2(triple)- and scAAV8(Y733F)- injected animals.
<p>GFP expression was detected in (A) scAAV(triple) and (B) scAAV8(Y733F)- injected mouse as well as in (C) scAAV2(triple)- and (D) scAAV8(Y733F)- injected rat corneas. Arrows indicate GFP- positive keratinocytes.</p
GFP expression in scAAV-injected mouse eyes.
<p>In A-D, representative sections of the region around the chamber angle are shown. The small inserts illustrate the cornea, the iris and the NPE. Nuclei are stained with DAPI. Arrows indicate GFP signals in the cornea, the iris, the chamber angle, the ciliary body and/or the NPE. Arrowheads mark the region of the TM.</p
Localization of the GFP signal in the anterior section of eyes injected with similar titers of scAAVs.
<p>The transduction efficiency (grading:-,-/+, +, ++) is based on the intensity and distribution of the GFP signal in tissues of the anterior section. Unless otherwise noted (see asterisks) the term cornea represents GFP-expression in the corneal endothelium.</p><p>* One out of five showed GFP-positivity in the corneal stroma in addition to the corneal endothelium.</p><p>** One out of five showed GFP-positivity in the corneal stroma.</p><p>Localization of the GFP signal in the anterior section of eyes injected with similar titers of scAAVs.</p
Co-staining of GFP (green, arrows) and TSP-1 (red, arrowheads) in chamber angle of injected rat eyes.
<p>(A) scAAV2(Y444F) and (B) scAAV2(triple) mediate efficient transgene expression in the region of the trabecular meshwork. Nuclei are stained with DAPI. Asterisk indicates Schlemm‘s canal.</p
GFP expression in scAAV-injected rat eyes.
<p>In A-D, representative sections of the region around the chamber angle are shown. The small inserts illustrate the cornea, the iris and the NPE. Nuclei are stained with DAPI. Arrows indicate GFP signals in the cornea, the iris, the chamber angle and/or the NPE. Arrowheads mark the region of the TM.</p
Injection procedure in mice.
<p>(A) The cornea is punctured closely anterior to the iridocorneal angle. (B) Before vector administration, an air bubble is created to seal the puncture site. (C-D) scAAV or vehicle dyed with fluorescein is injected. The air bubble seals the puncture site and reflux is minimized. The air bubble is absorbed within 24 hrs.</p
Experimental groups of titer-matched scAAV-injected and mock-injected eyes.
<p>A sample size of 5 eyes per group was analyzed by immunohistochemistry for GFP-expression. The experimental protocol was performed in C57BL/6 mice and Sprague Dawley rats.</p><p>Experimental groups of titer-matched scAAV-injected and mock-injected eyes.</p
Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors
<div><p>Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction <i>in vivo</i>. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored <i>in vitro</i> using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.</p></div
The GCaMP‑R Family of Genetically Encoded Ratiometric Calcium Indicators
We report on GCaMP-Rs,
a new family of genetically encoded ratiometric
calcium indicators that extend the virtues of the GCaMP proteins to
ratiometric measurements. We have engineered a tandem construct of
calcium-dependent GCaMP and calcium-independent mCherry fluorescent
proteins. The tandem design assures that the two proteins localize
in the same cellular compartment(s) and facilitates pixelwise ratiometric
measurements; however, Förster resonance energy transfer (FRET)
between the fluorophores reduces brightness of the sensor by up to
half (depending on the GCaMP variant). To eliminate FRET, we introduced
a rigid α-helix, the ER/K helix, between GCaMP and mCherry.
Avoiding FRET significantly increases the brightness (notably, even
at low calcium concentrations), the signal-to-noise ratio, and the
dynamic range