Broad-spectrum virus reduction in red cell concentrates using INACTINE PEN110 chemistry.

BACKGROUND AND OBJECTIVES: The risk of transmission of blood-borne pathogens by transfusion is a persistent problem in medicine. To address this safety issue, INACTINE PEN110 chemistry is being utilized to develop a process for preparing pathogen-reduced red blood cell concentrates (RBCC). The purpose of this study was to characterize the virucidal effectiveness of the INACTINE PEN110 chemistry in full units of RBCC by using a panel of viruses with diverse properties in composition, size and shape. MATERIALS AND METHODS: The panel included four enveloped (bovine viral diarrhoea virus, pseudorabies virus, vesicular stomatitis Indiana virus and sindbis virus), six non-enveloped (porcine parvovirus, human adenovirus 2, reovirus 3, vesicular exanthema of swine virus, bluetongue virus, and foot and mouth disease virus) and cell-associated (human immunodeficiency) viruses. All viruses were individually spiked into CPD/AS-1, CP2D/AS-3 and CPD/AS-5 RBCC units and treated with 0.1% PEN110 (vol/vol) at 22 +/- 2 degrees C for up to 22 +/- 2 h. The PEN110 treatment reaction was stopped by chemical quenching, and residual virus was assayed. The cytotoxicity effect of PEN110-treated RBCC on indicator cells and the potential interference with the ability of the virus to infect indicator cells was determined and taken into consideration for calculating the virus-reduction factors, to avoid underestimation or overestimation of the virus reduction. RESULTS: The kinetics of inactivation for viruses spiked into CPD/AS-1, CP2D/AS-3 and CPD/AS-5 RBCC were equivalent. All viruses analysed in this study were reduced to the limit of detection of the assay. The reduction factors for the virus panel ranged from 4.2 to 7.5 log10/ml. CONCLUSIONS: The results from the study demonstrate for the first time that a pathogen-reduction technology for RBCC can achieve a broad-spectrum virucidal effect against both enveloped and non-enveloped viruses. The broad spectrum of virucidal activity of INACTINE PEN110, and equivalent kinetics of virus inactivation in RBCC prepared using different commercially available RBC storage solutions, demonstrate the robustness of this pathogen-reduction process.

Anti-beta s-ribozyme reduces beta s mRNA levels in transgenic mice: potential application to the gene therapy of sickle cell anemia.

Our current strategy for gene therapy of sickle cell anemia involves retroviral vectors capable of transducing "designer" globin genes that code for novel anti-sickling globins (while resisting digestion by a ribozyme), coupled with the expression of a hammerhead ribozyme that can selectively cleave the human beta s mRNA. In this report, we have tested in vivo an anti-beta s hammerhead ribozyme embedded within a cDNA coding for the luciferase reporter gene driven by the human beta-globin promoter and hyper-sensitive sites 3 and 4 of the locus control region. We have created mice transgenic for this luciferase-ribozyme construct and bred the ribozyme transgene into mice that were already transgenic for the human beta s gene. We then measured expression of the beta s transgene at the protein and RNA levels by HPLC and primer extension. The presence of the ribozyme was associated with a statistically significant reduction in the level of beta s mRNA in spleen stress reticulocytes (from 60.5 +/- 4.1% to 52.9 +/- 4.2%) and in the percentage of beta s globin chains in very young mice (from 44.5 +/- 0.6% to 40.8 +/- 0.7%). These results demonstrate that it is possible to decrease the concentration of beta s chains and mRNA with the help of a hammerhead ribozyme. While the enormous amount of globin mRNA in reticulocytes is a challenge for ribozyme technology, the exquisite dependence of the delay time for formation of Hb S nuclei on the concentration of Hb S in red blood cells suggests that even a modest reduction in Hb S concentration would have therapeutic value.