Fermentation and Expression of Biopolymer Conjugates Used in Gene Therapy Treatments

While gene therapy has been touted as an effective method to treat cancer, providing a safe and effective carrier molecule for the delivery of the nucleic acid remains a challenge to biomedical engineering. Recently, a new strategy for delivery has been described which employs a novel recombinant fusion consisting of a block copolymer of amino acids (biopolymer) and a targeting factor (like FGF2). The properties of the biopolymer which provide very capable and efficient targeted gene delivery unfortunately make for difficult expression in simple batch cultures, with typical yields approaching about 100 μg per liter of bacterial culture. This obstacle can not be overcome by peptide synthesis because of the construct length. In order to provide sufficient material to investigate one type of block copolymer and how it affects therapeutic DNA condensation, a combination of biochemical engineering techniques were used to express (KKKHHHHKKK)₆FGF2. This conjugate is useful for the potential treatment of breast cancer.

This presentation will describe our efforts to increase expression of this gene delivery material based on improved plasmid design and advanced cultivation techniques. The plasmid design, based on arabinose induction, was able to control the timing of the fusion to reduce toxicity effects associated with product accumulation. Different induction timings and feeding profiles were investigated using fed batch in which raw materials and/or inducer is metered into the reactor based on an algorithm and online monitoring. Both a high cell mass (OD of about 45) and product (5 - 10 mg product per liter) was accumulated by fed batch, indicating that we were able to eliminate some of the limiting factors in expression. After purification via immobilized metal affinity chromatography, the transfection efficiency of (KKKHHHHKKK)₆FGF2 was examined in cell culture.