Towards liver-directed gene therapy: Retrovirus-mediated gene transfer into human hepatocytes

Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases. One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo. We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures. Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either the LacZ reporter gene or the cDNA for rabbit LDL receptor. The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell. Cytochemical assays were used to detect expression of the recombinant derived proteins, E. coli β-galactosidase and rabbit LDL receptor. Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels. The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45540/1/11188_2005_Article_BF01233625.pd

Water based fractionation of particles

A feed of particles covering a wide range of particle sizes and densities was placed into a novel separator, referred to as the Reflux Classifier, and fractionated at a series of fluidization velocities. Double fractionation was then achieved when each product was sieved into narrow size fractions. For the coal particles used in this study, the ash % was determined for each of the samples. The ash % correlated strongly with particle density, and hence was a useful proxy for the particle density. A new Fractionation Algorithm was developed, which provided a method for estimating the feed size and density distribution from the Double Fractionation data. The cumulative yield % versus the cumulative ash % curves for each size fraction, and for the overall feed were then determined. The findings from the study were validated against accurate results obtained using the sink-float method. Excellent agreement with the curves obtained using the sink-float method was obtained. The sink-float method requires the use of heavy liquids; these are invariably toxic and expensive, hence a viable alternative is needed. The water based fractionation method, in conjunction with the Fractionation Algorithm, may prove to be a suitable substitute for the sink-float method. Precise details of the method and algorithm are therefore provided in order to permit the adoption of this new approach by other laboratories