AAV-mediated and pharmacological induction of Hsp70 expression stimulates survival of retinal ganglion cells following axonal injury.

We evaluated the effect of AAV2- and 17-AAG (17-N-allylamino-17-demethoxygeldanamycin)-mediated upregulation of Hsp70 expression on the survival of retinal ganglion cells (RGCs) injured by optic nerve crush (ONC). AAV2-Hsp70 expression in the retina was primarily observed in the ganglion cell layer. Approximately 75% of all transfected cells were RGCs. RGC survival in AAV2-Hsp70-injected animals was increased by an average of 110% 2 weeks after the axonal injury compared with the control. The increase in cell numbers was not even across the retinas with a maximum effect of approximately 306% observed in the inferior quadrant. 17-AAG-mediated induction of Hsp70 expression has been associated with cell protection in various models of neurodegenerative diseases. We show here that a single intravitreal injection of 17-AAG (0.2 ug ul(-1)) results in an increased survival of ONC-injured RGCs by approximately 49% compared with the vehicle-treated animals. Expression of Hsp70 in retinas of 17-AAG-treated animals was upregulated approximately by twofold compared with control animals. Our data support the idea that the upregulation of Hsp70 has a beneficial effect on the survival of injured RGCs, and the induction of this protein could be viewed as a potential neuroprotective strategy for optic neuropathies

Light hadron spectra and wave functions in quenched QCD with overlap quarks on a large lattice

A simulation of quenched QCD with the overlap Dirac operator has been completed using 100 Wilson gauge configurations at beta=6 on an 18^3x64 lattice. We present results for meson and baryon masses, meson final state "wave functions'' and other observables.Comment: 5 LaTeX pages (espcrc2.sty), 13 figures. Combined contributions by J.H., L.L. and C.R. at Lattice2004(spectrum), Fermilab, June 21-26, 200

Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus

Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the incubation time needed to attain significant levels of gene expression is often too long for some clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA, eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency of these two types of AAV vectors in the murine myocardium. Four day old CD-1 mice were injected with either of the two AAV constructs, both expressing GFP and packaged into the AAV1 capsid. The animals were held for 4, 6, 11 or 21 days, after which they were euthanized and their hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR quantification of AAV genome copies and for confocal microscopy. Although we observed similar numbers of AAV genomes at each of the different time points present in both the scAAV and the ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals injected with the scAAV, while little or no expression was observed with the ssAAV constructs until day 11. AAV transduction of murine myocardium is therefore significantly enhanced using scAAV constructs

rAAV2/5 Gene-Targeting to Rods: Dose-Dependent Efficiency and Complications Associated With Different Promoters

A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken β actin proximal promoter CBA were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor-specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, whereas the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 1011 vg ml–1 were used

Parabolic stable surfaces with constant mean curvature

We prove that if u is a bounded smooth function in the kernel of a nonnegative Schrodinger operator $-L=-(\Delta +q)$ on a parabolic Riemannian manifold M, then u is either identically zero or it has no zeros on M, and the linear space of such functions is 1-dimensional. We obtain consequences for orientable, complete stable surfaces with constant mean curvature $H\in\mathbb{R}$ in homogeneous spaces $\mathbb{E}(\kappa,\tau)$ with four dimensional isometry group. For instance, if M is an orientable, parabolic, complete immersed surface with constant mean curvature H in $\mathbb{H}^2\times\mathbb{R}$, then $|H|\leq 1/2$ and if equality holds, then M is either an entire graph or a vertical horocylinder.Comment: 15 pages, 1 figure. Minor changes have been incorporated (exchange finite capacity by parabolicity, and simplify the proof of Theorem 1)

Recombinant AAV-Mediated \u3cem\u3eBEST1\u3c/em\u3e Transfer to the Retinal Pigment Epithelium: Analysis of Serotype-Dependent Retinal Effects

Mutations in the BEST1 gene constitute an underlying cause of juvenile macular dystrophies, a group of retinal disorders commonly referred to as bestrophinopathies and usually diagnosed in early childhood or adolescence. The disease primarily affects macular and paramacular regions of the eye leading to major declines in central vision later in life. Currently, there is no cure or surgical management for BEST1-associated disorders. The recently characterized human disease counterpart, canine multifocal retinopathy (cmr), recapitulates a full spectrum of clinical and molecular features observed in human bestrophinopathies and offers a valuable model system for development and testing of therapeutic strategies. In this study, the specificity, efficiency and safety of rAAV-mediated transgene expression driven by the human VMD2 promoter were assessed in wild-type canine retinae. While the subretinal delivery of rAAV2/1 vector serotype was associated with cone damage in the retina when BEST1 and GFP were co-expressed, the rAAV2/2 vector serotype carrying either GFP reporter or BEST1 transgene under control of human VMD2 promoter was safe, and enabled specific transduction of the RPE cell monolayer that was stable for up to 6 months post injection. These encouraging studies with the rAAV2/2 vector lay the groundwork for development of gene augmentation therapy for human bestrophinopathies