Scott A. Loiler

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.

Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver

Human pancreatic islet cells and hepatocytes represent the two most likely target cells for genetic therapy of type I diabetes. However, limits to the efficiency of rAAV serotype 2 (rAAV2)-mediated gene transfer have been reported for both of these cell targets. Here we report that nonserotype 2 AAV capsids can mediate more efficient transduction of islet cells, with AAV1 being the most efficient serotype in murine islets, suggesting that receptor abundance could be limiting. In order to test this, we generated rAAV particles that display a ligand (ApoE) that targets the low-density lipoprotein receptor, which is present on both of these cell types. The rAAV/ApoE viruses greatly enhanced the efficiency of transduction of both islet cells ex vivo and murine hepatocytes in vivo when compared to native rAAV2 serotype (220- and four-fold, respectively). The use of receptor-targeted rAAV particles may circumvent the lower abundance of receptors on certain nonpermissive cell types.

Enhancing rAAV vector expression in the lung.

Despite favorable DNA transfer efficiency, gene expression from recombinant adeno‐associated virus (rAAV2) vectors in the lung has been variable in the context of cystic fibrosis (CF) gene therapy. This is due, in part, to the large size of the CF transmembrane regulator (CFTR)‐coding sequence which necessitates the use of compact endogenous promoter elements versus stronger exogenous promoters. We evaluated the possibility that gene expression from rAAV could be improved by using AAV capsid serotypes with greater tropism for the apical surface of airway cells (i.e. rAAV5 or rAAV1) and/or using strong promoters such as the cytomegalovirus (CMV) enhancer/chicken beta‐actin hybrid (Cβ) promoter. The relative activity of the CMV immediate‐early (CMVie) promoter, the Cβ promoter, and the Cβ promoter with a downstream woodchuck hepatitis virus post‐transcriptional regulatory element (wpre) were assessed in vitro and in vivo in C57\Bl6 mice using human alpha‐1 antitrypsin (hAAT) as a secreted reporter. In vivo, the Cβ‐AAT‐wpre group achieved maximum serum levels of 1.5 mg/ml of hAAT. AAV capsid serotypes were then compared in vivo utilizing the transcriptionally optimized CB‐wpre cassette in rAAV serotype 1, 2 or 5 capsids (rAAV1, rAAV2, and rAAV5), utilizing luciferase as a reporter to compare expression over a wide dynamic range. The pulmonary luciferase levels at 8 weeks were similar in rAAV5 and rAAV1 groups (2.9 × 106 relative light units (RLU)/g tissue and 2.7 × 106 RLU/g tissue, respectively), both of which were much higher than rAAV2. Although the advantage of rAAV5 over rAAV2 in the lung has already been described, the availability of another serotype (rAAV1) capable of efficient gene transfer in the lung could be useful. Copyright

Glucose-Responsive Expression of the Human Insulin Promoter in HepG2 Human Hepatoma Cells

Abstract: The concept of insulin production afforded by hepatic gene therapy retains promise as a potential therapy for type 1 diabetes, but the approach has been limited by the need for strict transgene regulation in response to fluctuating levels of both glucose and insulin. Furthermore, while hepatocytes contain various glucose‐responsive elements, they lack the appropriate regulated secretory system necessary for insulin release, thereby necessitating the requirement for transcriptional regulation of hepatic insulin production under the direction of a glucose‐responsive promoter. To address this, we have evaluated several glucose‐responsive promoters that may be used successfully for hepatic insulin production via recombinant adeno‐associated virus (rAAV) therapy. Our results suggest that the human insulin promoter represents a strong candidate as a robust, glucose‐responsive promoter for regulated hepatic insulin production.

Duplication of U3 Sequences in the Long Terminal Repeat of Mink Cell Focus-Inducing Viruses Generates Redundancies of Transcription Factor Binding Sites Important for the Induction of Thymomas

ABSTRACT The ability of mink cell focus-inducing (MCF) viruses to induce thymomas is determined, in part, by transcriptional enhancers in the U3 region of their long terminal repeats (LTRs). To elucidate sequence motifs important for enhancer function in vivo, we injected newborn mice with MCF 1dr (supF), a weakly pathogenic, molecularly tagged (supF) MCF virus containing only one copy of a sequence that is present as two copies (known as the directly repeated [DR] sequence) in the U3 region of MCF 247 and analyzed LTRs from supF-tagged proviruses in two resulting thymomas. Tagged proviruses integrated upstream and in the reverse transcriptional orientation relative to c-myc provided the focus of our studies. These proviruses are thought to contribute to thymoma induction by enhancer-mediated deregulation of c-myc expression. The U3 region in a tagged LTR in one thymoma was cloned and sequenced. Relative to MCF 1dr (supF), the cloned U3 region contained an insertion of 140 bp derived predominantly from the DR sequence of the injected virus. The inserted sequence contains predicted binding sites for transcription factors known to regulate the U3 regions of various murine leukemia viruses. Similar constellations of binding sites were duplicated in two proviral LTRs integrated upstream from c-myc in a second thymoma. We replaced the U3 sequences in an infectious molecular clone of MCF 247 with the cloned proviral U3 sequences from the first thymoma and generated an infectious chimeric virus, MCF ProEn. When injected into neonatal AKR mice, MCF ProEn was more pathogenic than the parental virus, MCF 1dr (supF), as evidenced by the more rapid onset and higher incidence of thymomas. Molecular analyses of the resultant thymomas indicated that the U3 region of MCF ProEn was genetically stable. These data suggest that the arrangement and/or redundancy of transcription factor binding sites generated by specific U3 sequence duplications are important to the biological events mediated by MCF proviruses integrated near c-myc that contribute to transformation.

Alternative capsid strategies for targeting recombinant AAV gene therapy

The advent of new serotypes of adeno-associated viruses (AAV) has expanded the tissue tropism and allowed clinically relevant numbers of cells to be transduced by rAAV vectors. The two most significant obstacles in the way of future gene therapies with AAV vectors are tissue/target cell specificity and sitel-specific integration. Other reviews contained in this issue will look at advances in site-specific integration. This review will focus on the latest developments in AAV vector tropism and tissue/ target cell specificity.