Qing Ruan

Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors

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 in vivo. 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 in vitro 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.

PEDF Regulates Vascular Permeability by a γ-Secretase-Mediated Pathway

Increased vascular permeability is an inciting event in many vascular complications including diabetic retinopathy. We have previously reported that pigment epithelium-derived factor (PEDF) is able to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis through a novel γ-secretase-dependent pathway. In this study, we asked whether inhibition of VEGF-induced permeability by PEDF is also γ-secretase-mediated and to dissect the potential mechanisms involved. Vascular permeability was assessed in vitro by measuring transendothelial resistance and paracellular permeability to dextran and in vivo by following leakage of intravenous FITC-labelled albumin into the retina in the presence or absence of VEGF and PEDF in varying combinations. Experiments were undertaken in the presence or absence of a γ-secretase inhibitor. To assess junctional integrity immunohistochemistry for the adherens junction (AJ) proteins, VE-cadherin and β-catenin, and the tight junction (TJ) protein, claudin-5 was undertaken using cultured cells and flat mount retinas. Protein expression and the association between AJ proteins, VEGF receptors (VEGFRs) and γ-secretase constituents were determined by immunoprecipitation and Western Blot analysis. In selected experiments the effect of hypoxia on junctional integrity was also assessed. PEDF inhibition of VEGF-induced permeability, both in cultured microvascular endothelial cell monolayers and in vivo in the mouse retinal vasculature, is mediated by γ-secretase. PEDF acted by a) preventing dissociation of AJ and TJ proteins and b) regulating both the association of VEGF receptors with AJ proteins and the subsequent phosphorylation of the AJ proteins, VE-cadherin and β-catenin. Association of γ-secretase with AJ proteins appears to be critical in the regulation of vascular permeability. Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins. In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.

αB-crystallin, an effector of unfolded protein response, confers anti-VEGF resistance to breast cancer via maintenance of intracrine VEGF in endothelial cells.

Effective inhibition of angiogenesis targeting the tumor endothelial cells requires identification of key cellular and molecular mechanisms associated with survival of vasculatures within the tumor microenvironment. Intracellular autocrine (intracrine) VEGF production by endothelial cells plays a critical role on the vasculature homeostasis. In vitro breast cancer cell–stimulated activation of the unfolded protein response (UPR) of the endothelial cells contributes to maintenance of the intracrine VEGF levels in the endothelial cells through the upregulation of a previous undescribed downstream effector- αB-crystallin (CRYAB). siRNA-mediated knockdown of two major UPR proteins—inositol requiring kinase 1 and ATF6, led to attenuated CRYAB expression of the endothelial cells. Finally, inhibition of CRYAB blocked the breast cancer cell–stimulated increase in the endogenous VEGF levels of the endothelial cells. A VEGF limited proteolysis assay further revealed that CRYAB protected VEGF for proteolytic degradation. Here, we report that the molecular chaperone-CRYAB was significantly increased and colocalized with tumor vessels in a breast cancer xenograft. Specifically, neutralization of VEGF induced higher levels of CRYAB expression in the endothelial cells cocultured with MDA-MB-231 or the breast cancer xenograft with a significant survival benefit. However, knockdown of CRYAB had a greater inhibitory effect on endothelial survival. These findings underscore the importance of defining a role for intracrine VEGF signaling in sustaining aberrant tumor angiogenesis and strongly implicate UPR/CRYAB as dichotomous parts of a crucial regulation pathway for maintaining intracrine VEGF signaling. Mol Cancer Res; 9(12); 1632–43. ©2011 AACR.

γ-Secretase and Presenilin Mediate Cleavage and Phosphorylation of Vascular Endothelial Growth Factor Receptor-1

Background: γ-Secretase regulates VEGFR1 signaling. Results: Transmembrane cleavage of VEGFR1 occurs at valine 767, and VE-PTP dephosphorylation of activated VEGFR1 requires full-length presenilin 1. Conclusion: γ-Secretase cleaves VEGFR1, and full-length presenilin is a critical adaptor molecule in the dephosphorylation of VEGFR1. Significance: A greater understanding of PEDF-mediated VEGFR1 signaling and the role of γ-secretase/presenilin in the regulation of angiogenesis is important for cell biology. We have reported previously that pigment epithelium-derived factor (PEDF) can, via γ-secretase-mediated events, inhibit VEGF-induced angiogenesis in microvascular endothelial cells by both (a) cleavage and intracellular translocation of a C-terminal fragment of VEGF receptor-1 (VEGFR1) and (b) inhibition of VEGF-induced phosphorylation of VEGFR1. Using site-direct mutagenesis and transfection of wild type and mutated receptors into endothelial cells, we showed that transmembrane cleavage of VEGFR1 occurs at valine 767 and that a switch from valine to alanine at this position prevented cleavage and formation of a VEGFR1 intracellular fragment. Using siRNA to selectively knock down protein-tyrosine phosphatases (PTPs) in endothelial cells, we demonstrated that vascular endothelial PTP is responsible for dephosphorylation of activated VEGFR1. PEDF up-regulation of full-length presenilin 1 (Fl.PS1) facilitated the association of vascular endothelial PTP and VEGFR1. Knockdown of Fl.PS1 prevented dephosphorylation of VEGFR1, whereas up-regulation of Fl.PS1 stimulated VEGFR1 dephosphorylation. Fl.PS1 associated with VEGFR1 within 15 min after PEDF treatment. In conclusion, we determined the PEDF-mediated events responsible for VEGFR1 signaling and identified full-length presenilin as a critical adaptor molecule in the dephosphorylation of VEGFR1. This greater understanding of the regulation of VEGFR1 signaling will help identify novel anti-VEGF therapeutic strategies.

Development of an anti-angiogenic therapeutic model combining scAAV2-delivered siRNAs and noninvasive photoacoustic imaging of tumor vasculature development

We aimed to develop an anti-angiogenic model for breast cancer by combining (1) siRNA-based therapy delivered by self-complementary adeno-associated virus serotype 2 (scAAV2) vectors to target tumor vasculature, and (2) noninvasive monitoring to tumor response to anti-angiogenesis by photoacoustic (PA) imaging. scAAV2 vector containing 7 surface exposed tyrosine to phenylanine capsid mutations was able to transduce microvascular endothelial cells with high efficiency. siRNAs against UPR (unfolded protein response)-IRE1α, XBP-1, ATF6 significantly inhibited breast cancer-induced angiogenesis in vitro by inhibiting endothelial cell survival. PA imaging showed that knockdown of UPR proteins greatly reduced tumor angiogenesis in vivo in breast cancer models.

Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors.

ABSTRACT Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection. IMPORTANCE AAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors that can mediate transduction following noninvasive, intravitreal injection. HS binding has been postulated to play a role in intravitreally mediated transduction of retina. Our evaluation of the HS binding of AAV2-based variants and other AAV serotype vectors and the correlation of this property with transduction points to HS affinity as a factor controlling retinal transduction following Ivt delivery. However, HS binding is not the only requirement for improved Ivt-mediated transduction. We show that AAV2-based vectors lacking heparin binding transduce retina by subretinal injection and display a remarkable ability to transduce photoreceptors, indicating that other receptors are involved in this phenotype.

Free Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) Reduces Retinal Vascular Permeability in Association with a Reduction of Acid Sphingomyelinase (ASMase)

PURPOSE To examine the effect of free insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), independent of the effect of insulin-like growth factors, in modulating retinal vascular permeability. METHODS We assessed the ability of a form of IGFBP-3 that does not bind IGF-1 (IGFBP-3NB), to regulate the blood retinal barrier (BRB) using two distinct experimental mouse models, laser-induced retinal vessel injury and vascular endothelial growth factor (VEGF)-induced retinal vascular permeability. Additionally, in vitro studies were conducted. In the animal models, BRB permeability was quantified by intravenous injection of fluorescein labeled serum albumin followed by digital confocal image analysis of retinal flat-mounts. Claudin-5 and vascular endothelial-cadherin (VE-cadherin) localization at interendothelial junctions was studied by immunofluorescence. In vitro changes in transendothelial electrical resistance (TEER) and flux of fluorescent dextran in bovine retinal endothelial monolayers (BREC) were measured after IGFBP-3NB treatment. Acid (ASMase) and neutral (NSMase) sphingomyelinase mRNA levels and activity were measured in mouse retinas. RESULTS Four days postinjury, laser-injured mouse retinas injected with IGFBP-3NB plasmid demonstrated reduced vascular permeability compared with retinas of laser-injured mouse retinas injected with control plasmid. IGFBP-3NB administration resulted in a significant decrease in laser injury-associated increases in ASMase and NSMase mRNA and activity when compared with laser alone treated mice. In vivo, intravitreal injection of IGFBP-3NB reduced vascular leakage associated with intravitreal VEGF injection. IGFBP-3NB partially restored VEGF-induced in vivo permeability and dissociation of claudin-5 and VE-cadherin at junctional complexes. When IGFBP-3NB was applied basally to bovine retinal endothelial cells (BREC) in vitro, TEER increased and macromolecular flux decreased. CONCLUSIONS Intravitreal administration of IGFBP-3NB preserves junctional integrity in the presence of VEGF or laser injury by reducing BRB permeability in part by modulating sphingomyelinase levels.

Discovery of structure-based small molecular inhibitor of αB-crystallin against basal-like/triple-negative breast cancer development in vitro and in vivo.

AbstractαB-crystallin (CRYAB) is present at a high frequency in poor prognosis basal-like breast tumours, which are largely absent of oestrogen, progesterone receptors and HER2 known as triple-negative breast cancer (TNBC). CRYAB functions as a molecular chaperone to bind to and correct intracellular misfolded/unfolded proteins such as vascular endothelial growth factor (VEGF), preventing non-specific protein aggregations under the influence of the tumour microenvironment stress and/or anti-cancer treatments including bevacizumab therapy. Directly targeting CRYAB can sensitize tumour cells to chemotherapeutic agents and decrease tumour aggressiveness. However, growing evidence shows that CRYAB is a critical adaptive response element after ischemic heart disease and stroke, implying that directly targeting CRYAB might cause serious unwanted side effects. Here, we used structure-based molecular docking of CRYAB and identified a potent small molecular inhibitor, NCI-41356, which can strongly block the interaction between CRYAB and VEGF165 without affecting CRYAB levels. The disruption of the interaction between CRYAB and VEGF165 elicits in vitro anti-tumour cell proliferation and invasive effects through the down-regulation of VEGF signalling in the breast cancer cells. The observed in vitro anti-tumour angiogenesis of endothelial cells might be attributed to the down-regulation of paracrine VEGF signalling in the breast cancer cells after treatment with NCI-41356. Intraperitoneal injection of NCI-41356 greatly inhibits the tumour growth and vasculature development in in vivo human breast cancer xenograft models. Our findings provide ‘proof-of-concept’ for the development of highly specific structure-based alternative targeted therapy for the prevention and/or treatment of TNBC.

γ-Secretase Inhibition of Murine Choroidal Neovascularization Is Associated with Reduction of Superoxide and Proinflammatory Cytokines

PURPOSE This study aimed to determine whether upregulation of γ-secretase could inhibit laser-induced choroidal neovascularization (CNV) and if this was associated with a reduction in both oxidative stress and proinflammatory cytokines. METHODS γ-Secretase, or its catalytic subunit presenilin 1 (PS1), were upregulated by exposure to either pigment epithelial derived factor (PEDF) or an AAV2 vector containing a PS1 gene driven by a vascular endothelial-cadherin promoter. Retinal endothelial cells were infected with AAV2 or exposed to PEDF in the presence or absence of VEGF and in vitro angiogenesis determined. Mouse eyes either received intravitreal injection of PEDF, DAPT (a γ-secretase inhibitor) or PEDF + DAPT at the time of laser injury, or AAV2 infection 3 weeks before receiving laser burns. Lesion volume was determined 14 days post laser injury. Superoxide generation, antioxidant activity and the production of proinflammatory mediators were assessed. Knockdown of γ-secretase was achieved using siRNA. RESULTS γ-Secretase upregulation and PS1 overexpression suppressed VEGF-induced in vitro angiogenesis and in vivo laser-induced CNV. This was associated with a reduction in the expression of VEGF and angiogenin 1 together with reduced superoxide anion generation and an increase in MnSOD compared with untreated CNV eyes. PS1 overexpression reduced proinflammatory factors and microglial activation in eyes with CNV compared with control. siRNA inhibition of γ-secretase resulted in increased angiogenesis. CONCLUSIONS γ-Secretase, and in particular PS1 alone, are potent regulators of angiogenesis and this is due in part to stabilizing endogenous superoxide generation and reducing proinflammatory cytokine expression during CNV.

Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat

Background Adeno associated virus (AAV) is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV’s ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC) of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc) genomes in the anterior segment of the eye. Methodology/Principle Findings AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE), iris and chamber angle including trabecular meshwork, with scAAV2(Y444F) and scAAV2(triple) being the most efficient. Conclusions/Significance This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene-based therapies for glaucoma and acquired or inherited corneal anomalies.