Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs.

An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy.

Non-steroidal anti-inflammatory drugs and apoptosis in the gastrointestinal tract: potential role of the pentose phosphate pathways.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely prescribed drugs, primarily for treatment of arthritis. NSAIDs can have two effects independent of their anti-inflammatory action. In the stomach and small bowel long term NSAID consumption can lead to ulceration, whereas in the colon NSAID use can regress existing tumours. In this review, we hypothesise that NSAID-induced damage occurs predominantly by promoting apoptosis, involving a number of mechanisms depending on the type and the redox state of the cell. In addition to inhibiting cyclooxygenase (COX) activity, this includes interfering with glucose metabolism through both arms of the pentose phosphate pathways and energy production via glycolysis and oxidative phosphorylation. Shifting the cellular balance from proliferation to apoptosis is probably the most important outcome by which NSAIDs exhibit their differing actions. Understanding how these different pathways can be reconciled and their contribution to the balance between cell birth and cell death is the challenge for the future. The pentose phosphate pathways may provide a pivotal point for understanding links between factors which alter proliferative activity (e.g. COXs), provide energy metabolism (particularly aerobic and anaerobic metabolism of glucose), and change the redox state of the cell leading to apoptosis.

An orally administered growth factor extract derived from bovine whey suppresses breath ethane in colitic rats.

BACKGROUND: Lipid peroxidation is a potential mechanism of bowel damage in colitis. The effect of oral consumption of a bovine whey-derived growth factor extract (WGFE) on lipid peroxidation was assessed using the ethane breath test in the dextran sulphate sodium (DSS) model of ulcerative colitis (UC) in rats. METHODS: Groups of rats consumed water (control), 2% DSS in drinking water, 2% DSS with a WGFE-supplemented diet, or 2% DSS plus prednisolone (1 mg x kg(-1) x day(-1)) for 6 weeks, changing to sulphasalazine (100 mg x kg(-1) x day(-1)) for the subsequent 4 weeks. Ethane breath tests were conducted on all animals on days 2, 4, 6, 8, and 10 (acute phase) and weeks 3, 6, and 9 (chronic phase) after commencement of DSS consumption. RESULTS: There were no significant differences in ethane production between any groups during the acute phase. Ethane was significantly increased (P < 0.05) in rats consuming DSS alone in week 6 compared with control but had decreased to control levels by week 9. WGFE and conventional therapy were effective in suppressing ethane production in week 3. CONCLUSIONS: WGFE is as effective as conventional therapies at limiting ethane production and thus ostensibly colonic lipid peroxidation in the early phases of experimental chronic UC.