Identification of Novel Macropinocytosing Human Antibodies by Phage Display and High-Content Analysis.

Internalizing antibodies have great potential for the development of targeted therapeutics. Antibodies that internalize via the macropinocytosis pathway are particularly promising since macropinocytosis is capable of mediating rapid, bulk uptake and is selectively upregulated in many cancers. We hereby describe a method for identifying antibodies that internalize via macropinocytosis by screening phage-displayed single-chain antibody selection outputs with an automated fluorescent microscopy-based high-content analysis platform. Furthermore, this method can be similarly applied to other endocytic pathways if other fluorescent, pathway-specific, soluble markers are available.

Sustained correction of bleeding disorder in hemophilia B mice by gene therapy.

Mice generated by disrupting the clotting factor IX gene exhibit severe bleeding disorder and closely resemble the phenotype seen in hemophilia B patients. Here we demonstrate that a single intraportal injection of a recombinant adeno-associated virus (AAV) vector encoding canine factor IX cDNA under the control of a liver-specific enhancer/promoter leads to a long-term and complete correction of the bleeding disorder. High level expression of up to 15-20 microgram/ml of canine factor IX was detected in the plasma of mice injected with 5.6 x 10(11) particles of an AAV vector for >5 months. The activated partial thromboplastin time of the treated mice was fully corrected to higher than normal levels. Liver-specific expression of canine factor IX was confirmed by immunofluorescence staining, and secreted factor IX protein was identified in the mouse plasma by Western blotting. All treated mice survived the tail clip test without difficulty. Thus, a single intraportal injection of a recombinant adeno-associated virus vector expressing factor IX successfully cured the bleeding disorder of hemophilia B mice, proving the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.

Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A.

While the gene delivery vehicle is critical for the efficacy of human factor VIII gene therapy, optimization of the potency and duration of the factor VIII gene that is delivered is equally important in light of the poor transcription and translation characteristics of this gene. We discuss here a systematic approach to optimization of factor VIII complementary DNA expression by analysis of specific elements engineered into the transcription unit and other positions in the expression plasmid. Within the transcription unit we have engineered different 5' and 3' sequence modifications and tested them for factor VIII expression in human liver cells. These changes incorporate liver-specific promoter and enhancer sequences and regulatory elements affecting RNA export. Specifically, the thyroid hormone-binding globulin promoter and alpha 1 microglobulin/bikunin enhancer were tested and a synthetic 5' intron was compared to a 3' post-transcriptional regulatory element on factor VIII expression levels. For translation optimization, a leader sequence was designed to be of optimum length, have no RNA secondary structure and contain the optimal translation initiation sequence. Finally, we discuss areas for plasmid optimization, which include removal of near-consensus splicing sequences, the inclusion of strong transcription termination elements and the use of autonomous replicating plasmid sequences for episomal maintenance and enhanced plasmid retention for duration of gene expression.