AAV-mediated expression of HLA-G for the prevention of experimental ocular graft vs. host disease.

Ocular graft versus host disease (OGvHD) develops after allogeneic hematopoietic stem cell transplantation (HSCT) and manifests as ocular surface inflammatory disease. This study evaluated the efficacy of adeno-associated virus (AAV) gene therapy encoding human leukocyte antigen G (HLA-G) to inhibit OGvHD. A major histocompatibility mismatch chronic OGvHD murine model was evaluated. 7 days after HSCT, mice were dosed subconjunctivally with scAAV8-HLA-G1/5 (1 x 109 vg/eye), topical cyclosporine (twice daily), or left untreated. Body weights and tear production (red thread test) were recorded, and eyelid, corneal opacity, and corneal fluorescein retention were scored through day 44 after HSCT. Tissues were collected for vector biodistribution, ocular histology, and immunofluorescence. Compared with untreated HSCT eyes, those dosed with scAAV8-HLA-G1/5 had significantly reduced clinical inflammatory signs of OGvHD. On histology, eyes that received scAAV8-HLA-G1/5 or cyclosporine had a significantly lower mean limbal mononuclear cell count when compared with non-treated HSCT eyes. HLA-G immunofluorescence was detected in the subconjunctiva and peripheral cornea in HSCT animals treated with scAAV8-HLA-G1/5. Vector genomes were detected in the lacrimal gland, but not in the other tested organs. These results provide evidence that subconjunctival AAV targets ocular surface and corneal disease and support that HLA-G-based gene therapy may be an effective treatment for OGvHD.

Inhibition of experimental autoimmune uveitis by intravitreal AAV-Equine-IL10 gene therapy.

Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy for inhibition of experimental autoimmune uveitis (EAU) in rats. Each rat was dosed intravitreally (IVT) in both eyes with either balanced salt solution (BSS) (control; n = 4), AAV8-Equine-IL10 at a low dose (2.4x109 vg; n = 5) or high dose (2.4x1010 vg; n = 5). EAU was induced in all groups of rats 7 days after IVT injections and euthanized 21 days post-injection. Ophthalmic examination and aqueous humor (AH) cell counts were recorded with the observer blinded to the treatment groups. Histopathology and qPCR were performed on selected ocular tissues. Data presented herein demonstrate that AAV8-Equine-IL10 treated rats exhibited a significant decrease in clinical inflammatory scores and AH cell counts compared to BSS-treated EAU eyes on days 10, 12 and 14 post EAU induction at both administered vector doses. Mean cellular histologic infiltrative scores were also significantly less in AAV8-Equine-IL10 dosed rats compared to the BSS group. Intravitreal injection of AAV8-Equine-IL10 resulted in Equine-IL10 cDNA expression in the ciliary body, retina, cornea, and optic nerve in a dose-dependent manner. A single IVT injection of AAV8-Equine-IL10 appeared to be well-tolerated and inhibited EAU even at the lowest administered dose. These results demonstrate safety and efficacy of AAV8-Equine-IL10 to prevent EAU and support continued exploration of AAV gene therapy for the treatment of equine and perhaps human recurrent uveitis.

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models.

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SC) injections offer advantages over other intraocular administration routes as they are simple, safe, require only local anesthesia, and are usually performed in an outpatient setting. Although SC injections in small animals usually require the assistance of an operating microscope due to the size of the eye, they are widely utilized for drug delivery, including gene therapy vectors. Previous work has demonstrated that SC injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases. Herein, a detailed protocol is presented for SC injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

Harnessing the Natural Biology of Adeno-Associated Virus to Enhance the Efficacy of Cancer Gene Therapy.

Adeno-associated virus (AAV) was first characterized as small "defective" contaminant particles in a simian adenovirus preparation in 1965. Since then, a recombinant platform of AAV (rAAV) has become one of the leading candidates for gene therapy applications resulting in two FDA-approved treatments for rare monogenic diseases and many more currently in various phases of the pharmaceutical development pipeline. Herein, we summarize rAAV approaches for the treatment of diverse types of cancers and highlight the natural anti-oncogenic effects of wild-type AAV (wtAAV), including interactions with the cellular host machinery, that are of relevance to enhance current treatment strategies for cancer.

Ocular Tolerability and Immune Response to Corneal Intrastromal AAV-IDUA Gene Therapy in New Zealand White Rabbits.

The chronic ocular toxicity, tolerability, and inflammation following corneal intrastromal injection of saline or escalating doses of an adeno-associated virus (AAV) containing a codon-optimized α-l-iduronidase (AAV-opt-IDUA) expression cassette were evaluated in New Zealand White rabbits. Corneal opacity following corneal intrastromal injection resolved by 24 h. Mild elevation of clinical ocular inflammation was observed 24 h after injection, but it returned to baseline by day 7 and no abnormalities were noted through 6 months of observation after injection. Vector genomes and IDUA cDNA were detected in the injected corneas in a dose-dependent manner. Both the lowest administered AAV-opt-IDUA dose, shown to be effective in mucopolysaccharidosis type I (MPS I) dogs, and a 10-fold higher dose of AAV-opt-IDUA resulted in no detectable immunologic response or adverse effect in rabbits. Vector genomes outside of the eye were rarely detected following corneal intrastromal injection of AAV-opt-IDUA, and neutralizing antibodies to the AAV capsid were not present at the experimental conclusion. This study, combined with our previous studies in MPS I dogs, suggests that AAV-opt-IDUA corneal gene therapy following corneal intrastromal injection of AAV-opt-IDUA has the potential to prevent and reverse blindness in MPS I patients in a safe and effective manner.

Preparation and Administration of Adeno-associated Virus Vectors for Corneal Gene Delivery.

Gene delivery approaches using adeno-associated virus (AAV) vectors are currently the preferred method for human gene therapy applications and have demonstrated success in clinical trials for a diverse set of diseases including retinal blindness. To date, no clinical trials using AAV gene therapy in the anterior eye have been initiated; however, corneal gene delivery appears to be an attractive approach for treating both corneal and ocular surface diseases. Multiple preclinical studies by our lab and others have demonstrated efficient AAV vector-mediated gene delivery to the cornea for immunomodulation, anti-vascularization, and enzyme supplementation. Interestingly, the route of AAV vector administration and nuances such as administered volume influence vector tropism and transduction efficiency. In this chapter, a detailed protocol for AAV vector production and specific approaches for AAV-mediated gene transfer to the cornea via subconjunctival and intrastromal injections are described.

AAV-mediated expression of HLA-G1/5 reduces severity of experimental autoimmune uveitis.

Non-infectious uveitis (NIU) is an intractable, recurrent, and painful disease that is a common cause of vision loss. Available treatments of NIU, such as the use of topical corticosteroids, are non-specific and have serious side effects which limits them to short-term use; however, NIU requires long-term treatment to prevent vision loss. Therefore, a single dose therapeutic that mediates long-term immunosuppression with minimal side effects is desirable. In order to develop an effective long-term therapy for NIU, an adeno-associated virus (AAV) gene therapy approach was used to exploit a natural immune tolerance mechanism induced by the human leukocyte antigen G (HLA-G). To mimic the prevention of NIU, naïve Lewis rats received a single intravitreal injection of AAV particles harboring codon-optimized cDNAs encoding HLA-G1 and HLA-G5 isoforms one week prior to the induction of experimental autoimmune uveitis (EAU). AAV-mediated expression of the HLA-G-1 and -5 transgenes in the targeted ocular tissues following a single intravitreal injection of AAV-HLA-G1/5 significantly decreased clinical and histopathological inflammation scores compared to untreated EAU eyes (p < 0.04). Thus, localized ocular gene delivery of AAV-HLA-G1/5 may reduce the off-target risks and establish a long-term immunosuppressive effect that would serve as an effective and novel therapeutic strategy for NIU, with the potential for applications to additional ocular immune-mediated diseases.

Patterns of cell cycle checkpoint deregulation associated with intrinsic molecular subtypes of human breast cancer cells.

Genomic instability is a hallmark of breast cancer, contributes to tumor heterogeneity, and influences chemotherapy resistance. Although Gap 2 and mitotic checkpoints are thought to prevent genomic instability, the role of these checkpoints in breast cancer is poorly understood. Here, we assess the Gap 2 and mitotic checkpoint functions of 24 breast cancer and immortalized mammary epithelial cell lines representing four of the six intrinsic molecular subtypes of breast cancer. We found that patterns of cell cycle checkpoint deregulation were associated with the intrinsic molecular subtype of breast cancer cell lines. Specifically, the luminal B and basal-like cell lines harbored two molecularly distinct Gap 2/mitosis checkpoint defects (impairment of the decatenation Gap 2 checkpoint and the spindle assembly checkpoint, respectively). All subtypes of breast cancer cell lines examined displayed aberrant DNA synthesis/Gap 2/mitosis progression and the basal-like and claudin-low cell lines exhibited increased percentages of chromatid cohesion defects. Furthermore, a decatenation Gap 2 checkpoint gene expression signature identified in the cell line panel correlated with clinical outcomes in breast cancer patients, suggesting that breast tumors may also harbor defects in decatenation Gap 2 checkpoint function. Taken together, these data imply that pharmacological targeting of signaling pathways driving these phenotypes may lead to the development of novel personalized treatment strategies for the latter two subtypes which currently lack targeted therapeutic options because of their triple negative breast cancer status.

Human embryonic stem cells have constitutively active Bax at the Golgi and are primed to undergo rapid apoptosis.

Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax, which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having preactivated Bax. After DNA damage, active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly, upon differentiation, Bax was no longer active, and cells were not acutely sensitive to DNA damage. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development.

Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

Revised genetic requirements for the decatenation G2 checkpoint: the role of ATM.

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.

As(III) transcriptionally activates the gadd45a gene via the formation of H2O2.

Arsenic is a ubiquitous environmental contaminant associated with increased risks of human cancers of the skin, lung, bladder, and prostate. Intriguingly, it is also used to treat certain types of leukemia. It has recently been suggested that these paradoxic effects may be mediated by arsenic's ability to simultaneously activate DNA damage and apoptotic and transformation pathways. Here, we investigate the effects of arsenic exposure on the induction of the growth arrest and DNA damage protein 45 alpha (GADD45 alpha), which is thought to play roles in apoptosis, DNA damage response, and cell cycle arrest. We found that arsenic transcriptionally activates the gadd45 alpha promoter located in a 153-bp region between -234 and -81, relative to the transcriptional start site. In addition, this transcriptional induction was abrogated in the presence of H2O2 scavengers, suggesting a role for H2O2 in the transcriptional control of the gadd45a gene through a Fenton-like free radical mechanism.

Conference overview: molecular mechanisms of metal toxicity and carcinogenesis.

Chronic exposure to many heavy metals and metal-derivatives is associated with an increased risk of cancer, although the mechanisms of tumorigenesis are largely unknown. Approximately 125 scientists attended the 3rd Conference on Molecular Mechanisms of Metal Toxicity and Carcinogenesis and presented the latest research concerning these mechanisms. Major areas of focus included exposure assessment and biomarker identification, roles of ROS and antioxidants in carcinogenesis, mechanisms of metal-induced DNA damage, metal signalling, and the development of animal models for use in metal toxicology studies. Here we highlight some of the research presented, and summarize the conference proceedings.

Environmental health research in the post-genome era: new fields, new challenges, and new opportunities.

The human genome sequence provides researchers with a genetic framework to eventually understand the relationships of gene-environment interactions. This wealth of information has led to the birth of several related areas of research, including proteomics, functional genomics, pharmacogenomics, and toxicogenomics. Developing techniques such as DNA/protein microarrays, small-interfering RNA (siRNA) applications, two-dimensional gel electrophoresis, and mass spectrometry in conjunction with advanced analysis software and the availability of Internet databases offers a powerful set of tools to investigate an individual's response to specific stimuli. This review summarizes these emerging scientific fields and techniques focusing specifically on their applications to the complexities of gene-environment interactions and their potential role in environ-mental biosecurity.

Metal-induced toxicity, carcinogenesis, mechanisms and cellular responses.

A wide variety of metals have been reported to act as mutagenic and carcinogenic agents in both human and animal studies. The underlying mechanisms are being extensively investigated. Recently, a new sub-discipline of molecular carcinogenesis has surfaced and new techniques and instruments are being developed which allow exploration of the complex biological relationships and signaling pathways involved in response to metal exposure at the molecular level. The 2nd Conference on Molecular Mechanisms of Metal Toxicity and Carcinogenesis was held at NIOSH in Morgantown, West Virginia, Sept. 8-11, 2002. One hundred thirty scientist from sixteen countries presented their novel findings and investigations of metal-induced carcinogenesis. The conference focused on state-of-the-art research and developments in metal toxicity and carcinogenesis. Emphasis was placed on delineating molecular mechanisms involved in free radical effects, cellular uptake, signaling pathways/interaction, dose response, biomarkers, and resistance mechanisms. This article reviews some of the novel information presented at the conference and discusses future avenues of research in this field.