Adeno-Associated Virus (AAV) as a Vector for Gene Therapy.

There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies. The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy.

Deep sequencing approaches to antibody discovery.

Antibodies have become one of the dominant therapeutic platforms due to their safety, specificity, and efficacy. Owing to their massive potential diversity intrinsic to their structure, the number of possible different molecules that could be generated and analyzed from natural or synthetic systems is almost limitless. However, even with vast improvements in automation, classic antibody generation and analysis systems are severely limited in the number of molecules that can be interrogated during a typical discovery campaign. When one considers more complex target systems, along with the desire to isolate antibodies with very unique characteristics, the chances are very low that these systems will be successful. Next generation sequencing technologies (also referred to as "deep sequencing") allow for the analysis of single molecules in millions in a very short period of time. By applying these deep sequencing technologies to antibody discovery, we now have the ability to look for very specific molecules with very unique properties and activities, further our understanding of species and strain specific repertoires, and can now begin to use sequence information to identify function. The application of these technologies is opening the door to the discovery of next generation antibody therapeutics.

Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery.

Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.

Distinct roles of IL-23 and IL-17 in the development of psoriasis-like lesions in a mouse model.

Psoriasis is an inflammatory disease with dynamic interactions between the immune system and the skin. The IL-23/Th17 axis plays an important role in the pathogenesis of psoriasis, although the exact contributions of IL-23 and IL-17 in vivo remain unclear. K5.Stat3C transgenic mice constitutively express activated Stat3 within keratinocytes, and these animals develop skin lesions with histological and cytokine profiles similar to those of human plaque psoriasis. In this study, we characterized the effects of anti-mouse IL-17A, anti-mouse IL-12/23p40, and anti-mouse IL-23p19 Abs on the development of psoriasis-like lesions in K5.Stat3C transgenic mice. Treatment with anti-IL-12/23p40 or anti-IL-23p19 Abs greatly inhibited 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia in the ears of K5.Stat3C mice, whereas the inhibitory effect of an anti-IL-17A Ab was relatively less prominent. Treatment with anti-IL-12/23p40 or anti-IL-23p19 Abs markedly lowered transcript levels of Th17 cytokines (e.g., IL-17 and IL-22), ß-defensins, and S100A family members in skin lesions. However, anti-IL-17A Ab treatment did not affect mRNA levels of Th17 cytokines. Crossing IL-17A-deficient mice with K5.Stat3C mice resulted in partial attenuation of 12-O-tetradecanoylphorbol-13-acetate-induced lesions, which were further attenuated by anti-IL-12/23p40 Ab treatment. FACS analysis of skin-draining lymph node cells from mice that were intradermally injected with IL-23 revealed an increase in both IL-22-producing T cells and NK-22 cells. Taken together, this system provides a useful mouse model for psoriasis and demonstrates distinct roles for IL-23 and IL-17.

Engineering host cell lines to reduce terminal sialylation of secreted antibodies.

Covalently-linked glycans on proteins have many functional roles, some of which are still not completely understood. Antibodies have a very specific glycan modification in the Fc region that is required for mediating immune effector functions. These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is influenced by many factors, such as type of cellular host and rate of Ab secretion. Glycan heterogeneity can affect the Fc-dependent activities of antibodies. It has been shown recently that increased Fc sialylation can result in decreased binding to immobilized antigens and some Fcγ receptors, as well as decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of antibodies. To produce antibodies with increased effector functions, we developed host cell lines that would limit the degree of sialylation of recombinantly-expressed antibodies. Towards this end, the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) was engineered for secreted expression in mammalian cell lines. Expression of this sialidase A gene in mammalian cells resulted in secreted expression of soluble enzyme that was capable of removing sialic acid from antibodies secreted into the medium. Purified antibodies secreted from these cells were found to possess very low levels of sialylation compared with the same antibodies purified from unmodified host cells. The low sialylated antibodies exhibited similar binding affinity to soluble antigens, improved ADCC activity, and they possessed pharmacokinetic properties comparable to their more sialylated counterparts. Further, it was observed that the amount of sialidase A expressed was sufficient to thoroughly remove sialic acid from Abs made in high-producing cell lines. Thus, engineering host cells to express sialidase A enzyme can be used to produce recombinant antibodies with very low levels of sialylation.

Expression and characterization of a human BMP-7 variant with improved biochemical properties.

Bone morphogenetic protein-7 (BMP-7, OP-1) is a secreted growth factor that is predominantly known for its osteoinductive properties, though it has also been implicated as having a role in mammalian kidney development. Clinical efficacy of recombinant BMP-7 has been demonstrated in the treatment of orthopedic injuries through topical application. However, the pharmaceutical development of recombinant BMP-7 for systemic delivery has presented many challenges. Specifically, the expression level of recombinant mature BMP-7 protein in mammalian cells is very low, the molecule has poor solubility at neutral pH, and intracellular proteolytic processing events result in a secreted BMP-7 having multiple amino-termini, creating a heterogeneous mixture of proteins. Utilizing structural information, we have designed and generated a number of rational BMP-7 mutations that improved both expression levels in mammalian cells and solubility at neutral pH, while limiting the amino-terminal heterogeneity of the mature protein. Introduction of these mutations did not compromise BMP-7 in vitro bioactivity. This improved BMP-7 molecule is better suited for pharmaceutical development and clinical advancement for indications where systemic delivery may be required.

Dermokine: an extensively differentially spliced gene expressed in epithelial cells.

Studies performed to discover genes overexpressed in inflammatory diseases identified dermokine as being upregulated in such disease conditions. Dermokine is a gene that was first observed as expressed in the differentiated layers of skin. Its two major isoforms, alpha and beta, are transcribed from different promoters of the same locus, with the alpha isoform representing the C terminus of the beta isoform. Recently, additional transcript variants have been identified. Extensive in silico analysis and reverse transcriptase (RT)-PCR cloning has confirmed the existence of these variants in human cells and tissues, identified a new human isoform as well as the gamma isoform in mouse. Recombinant expression and analysis of the C-terminal truncated isoform indicate that the molecule is O-linked glycosylated and forms multimers in solution. In situ hybridization and immunohistochemistry has shown that the gene is differentially expressed in various cells and tissues, other than the skin. These results show that the dermokine gene is expressed in epithelial tissues other than the skin and this expression is transcriptionally and posttranscriptionally complex.