Vivian W. Choi

Production and characterization of adeno-associated viral vectors

The adeno-associated virus (AAV) is one of the most promising viral vectors for human gene therapy. As with any potential therapeutic system, a thorough understanding of it at the in vitro and in vivo levels is required. Over the years, numerous methods have been developed to better characterize AAV vectors. These methods have paved the way to a better understanding of the vector and, ultimately, its use in clinical applications. This review provides an up-to-date, detailed description of essential methods such as production, purification and titering and their application to characterize current AAV vectors for preclinical and clinical use.

AAV hybrid serotypes: improved vectors for gene delivery.

In recent years, significant efforts have been made on studying and engineering adeno-associated virus (AAV) capsid, in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest. With our previous knowledge of the viral properties of the naturally occurring serotypes and the elucidation of their capsid structures, we can now generate capsid mutants, or hybrid serotypes, by various methods and strategies. In this review, we summarize the studies performed on AAV retargeting, and categorize the available hybrid serotypes to date, based on the type of modification: 1) transcapsidation, 2) adsorption of bi-specific antibody to capsid surface, 3) mosaic capsid, and 4) chimeric capsid. Not only these hybrid serotypes could achieve high efficiency of gene delivery to a specific targeted cell type, which can be better-tailored for a particular clinical application, but also serve as a tool for studying AAV biology such as receptor binding, trafficking and genome delivery into the nucleus.

Production of recombinant adeno-associated viral vectors.

Adeno‐associated virus is a nonpathogenic human virus that has been developed into a gene‐delivery vector due to its high efficiency of infection for many different cell types and its ability to persist and lead to long‐term gene expression. This unit describes efficient methods to generate high‐titer, research‐grade, adenovirus‐free recombinant single‐stranded and self‐complementary adeno‐associated virus in various serotypes, along with methods to quantify the viral vectors.

Self-complementary AAV virus (scAAV) safe and long-term gene transfer in the trabecular meshwork of living rats and monkeys.

PURPOSE AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals. METHODS Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays. RESULTS No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys. CONCLUSIONS The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.

Effects of Adeno-Associated Virus DNA Hairpin Structure on Recombination

ABSTRACT Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence.

Mechanisms of AAV transduction in glaucoma-associated human trabecular meshwork cells.

Glaucoma is a chronic eye disease which leads to irreversible blindness. The trabecular meshwork tissue controls intraocular pressure (IOP), which is the major risk factor for glaucoma. Gene therapy treatment of chronic diseases requires the use of long‐term expression, low toxicity and lack of immune response vectors. Adeno‐associated viruses (AAV) possess these characteristics but have been unable to transduce the trabecular meshwork. Because of the importance of regulating elevated IOP by long‐term gene therapy, we investigated mechanisms of AAV transduction to the human trabecular meshwork (TM).

Production of Recombinant Adeno‐Associated Viral Vectors for In Vitro and In Vivo Use

Adeno‐associated virus is a nonpathogenic human virus that has been developed into a gene‐delivery vector due to its high efficiency of infection in many different cell types and its ability to persist and lead to long‐term gene expression. The vector is also a valuable tool in molecular biology experiments. This unit describes efficient methods to generate high‐titer, research‐grade, adenovirus‐free, recombinant single‐stranded and self‐complementary adeno‐associated virus in various serotypes, along with methods to quantify the viral vectors.

Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration

Adeno‐associated virus is a nonpathogenic human virus that has been developed into a gene‐delivery vector due to its high efficiency of infection for many different cell types and its ability to persist and lead to long‐term gene expression. This unit describes efficient methods to generate high‐titer, research‐grade, adenovirus‐free recombinant single‐stranded and self‐complementary adeno‐associated virus in various serotypes, along with methods to quantify the viral vectors. Two detailed methods are provided for viral vector delivery into the rodent brain and spinal cord, and for histological detection of transgene expression of GFP. Curr. Protoc. Neurosci. 57:4.17.1‐4.17.30.

VHL and PTEN loss coordinate to promote mouse liver vascular lesions

Von Hippel-Lindau (VHL) inactivation develops a tumor syndrome characterized by highly vascularized tumors as a result of hypoxia inducible factors (HIF) stabilization. The most common manifestation is the development of hemangioblastomas typically located in the central nervous system and other organs including the liver. PTEN (Phosphatase and tension homologue deleted on chromosome 10) inactivation also upregulates HIF-1α and may take part in promoting vascular lesions in tumors. The coordinate effect of loss of these tumor suppressors on HIF levels, and the subsequent effect on vascular lesion formation would elucidate the potential for mechanisms to modify HIF dosage supplementally and impact tumor phenotype. We therefore employed models of somatic conditional inactivation of Vhl, Pten, or both tumor suppressor genes in individual cells of the liver by Cre-loxP recombination to study the cooperativity of these two tumor suppressors in preventing tumor formation. Nine months after tumor suppressor inactivation, Vhl conditional deletion (VhlloxP/loxP) mice showed no abnormalities, Pten conditional deletion (PtenloxP/loxP) mice developed liver steatosis and focal nodular expansion of hepatocytes containing lipid droplet and fat. Vhl and Pten conditional deletion (VhlloxP/loxP;PtenloxP/loxP) mice, however, developed multiple cavernous liver lesions reminiscent of hemangioblastoma. Liver hemangioblastomas in VHL disease may, therefore, require secondary mutation in addition to VHL loss of heterozygosity which is permissive for vascular lesion development or augments levels of HIF-1α.

343. Repair of Thalassemic |[beta]|-Globin pre-mRNA by Antisense AAV and Lentiviral Vectors in Cell Culture

b-Thalassemia is a worldwide genetic blood disorder characterized by a deficiency or lack of the b-globin chain production. More than 200 mutations in the b-globin gene are responsible for this disease. Several common mutations create aberrant splice sites in the b-globin pre-mRNA, leading to an incorrectly spliced b-globin mRNA. Previous study in this lab showed that a modified U7.623 snRNA, which carries a specific sequence antisense to the splicing enhancer sequence of the three b-thalassemic mutants (IVS2-654, -705, and -745), could modify the splicing pathway and restore correctly spliced b-globin mRNA and protein in cell culture. In this study we incorporated the U7.623 antisense snRNA into lentiviral- and AAV-vectors. Transient transfection of HeLa cell lines expressing IVS2-654, and-705, with an AAV-U7 antisense snRNA plasmid, resulted in a restoration of both correctly spliced b-globin mRNA and full-length b-globin polypeptide. The results show that antisense snRNAs shifted the splicing pathway from aberrant to correct and restored the correct mRNAs in a sequence specific and dose-dependent manner. This was accomplished in model cell lines, in which the thalassemic b-globin intron was incorporated into the enhanced green fluorescence protein (EGFP) coding sequence and in HeLa cells that express thalassemic human b-globin genes. Transfection of EGFP-HeLa cell lines with IVS2-654, and -705U introns with AAV-U7.623 resulted in upregulation and expression of EGFP protein concomitant with expression of the of red fluorescence protein (RFP), an AAV viral reporter gene. Similar results, although less pronounced, were obtained in cells transiently transfected with lentiviral-U7.623 antisense snRNA construct. Interestingly, the restoration of correctly spliced mRNAs was faster in AAV than lentiviral vector treated cells. Based on this encouraging result, the AAV- and lentiviral-U7 antisense snRNA constructs will be evaluated in animal models for thalassemia. The snRNA antisense-based gene therapy may offer an attractive approach to cure thalassemia.