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.

Influence of particle size on solid solution formation and phase interfaces in Li0.5FePO4 revealed by 31P and 7Li solid state NMR spectroscopy.

Here we report the observation of electron delocalization in nano-dimension xLiFePO(4):(1 - x)FePO(4) (x = 0.5) using high temperature, static, (31)P solid state NMR. The (31)P paramagnetic shift in this material shows extreme sensitivity to the oxidation state of the Fe center. At room temperature two distinct (31)P resonances arising from FePO(4) and LiFePO(4) are observed at 5800 ppm and 3800 ppm, respectively. At temperatures near 400 °C these resonances coalesce into a single narrowed peak centered around 3200 ppm caused by the averaging of the electronic environments at the phosphate centers, resulting from the delocalization of the electrons among the iron centers. (7)Li MAS NMR spectra of nanometre sized xLiFePO(4):(1 - x)FePO(4) (x = 0.5) particles at ambient temperature reveal evidence of Li residing at the phase interface between the LiFePO(4) and FePO(4) domains. Moreover, a new broad resonance is resolved at 65 ppm, and is attributed to Li adjacent to the anti-site Fe defect. This information is considered in light of the (7)Li MAS spectrum of LiMnPO(4), which despite being iso-structural with LiFePO(4) yields a remarkably different (7)Li MAS spectrum due to the different electronic states of the paramagnetic centers. For LiMnPO(4) the higher (7)Li MAS paramagnetic shift (65 ppm) and narrowed isotropic resonance (FWHM ≈ 500 Hz) is attributed to an additional unpaired electron in the t(2g) orbital as compared to LiFePO(4) which has δ(iso) = -11 ppm and a FWHM = 9500 Hz. Only the delithiated phase FePO(4) is iso-electronic and iso-structural with LiMnPO(4). This similarity is readily observed in the (7)Li MAS spectrum of xLiFePO(4):(1 - x)FePO(4) (x = 0.5) where Li sitting near Fe in the 3+ oxidation state takes on spectral features reminiscent of LiMnPO(4). Overall, these spectral features allow for better understanding of the chemical and electrochemical (de)lithiation mechanisms of LiFePO(4) and the Li-environments generated upon cycling.

A multifunctional 3.5 V iron-based phosphate cathode for rechargeable batteries.

In the search for new positive-electrode materials for lithium-ion batteries, recent research has focused on nanostructured lithium transition-metal phosphates that exhibit desirable properties such as high energy storage capacity combined with electrochemical stability. Only one member of this class--the olivine LiFePO(4) (ref. 3)--has risen to prominence so far, owing to its other characteristics, which include low cost, low environmental impact and safety. These are critical for large-capacity systems such as plug-in hybrid electric vehicles. Nonetheless, olivine has some inherent shortcomings, including one-dimensional lithium-ion transport and a two-phase redox reaction that together limit the mobility of the phase boundary. Thus, nanocrystallites are key to enable fast rate behaviour. It has also been suggested that the long-term economic viability of large-scale Li-ion energy storage systems could be ultimately limited by global lithium reserves, although this remains speculative at present. (Current proven world reserves should be sufficient for the hybrid electric vehicle market, although plug-in hybrid electric vehicle and electric vehicle expansion would put considerable strain on resources and hence cost effectiveness.) Here, we report on a sodium/lithium iron phosphate, A(2)FePO(4)F (A=Na, Li), that could serve as a cathode in either Li-ion or Na-ion cells. Furthermore, it possesses facile two-dimensional pathways for Li+ transport, and the structural changes on reduction-oxidation are minimal. This results in a volume change of only 3.7% that--unlike the olivine--contributes to the absence of distinct two-phase behaviour during redox, and a reversible capacity that is 85% of theoretical.

Co, Fe and Ga chelates for cell labelling: a potential use in PET imaging?

The radiolabelling of blood cellular elements with PET radionuclides offers a higher sensitivity and resolution than conventional imaging, but short-lived PET radionuclides have limited clinical use in cell labelling. Medium half-life PET radionuclides, such as 55Co (t1/2 = 18.2 h), 52Fe (t1/2 = 8.3 h) and 66Ga (t1/2 = 9.4 h), enable quantitative uptake and cell kinetic studies with radiolabelled blood cellular elements. Co(II) oxine, Co(III) tropolonate, Fe(III) oxine, Ga(III) oxine and Ga(III) MPO were prepared using gamma-emitting radionuclides and a number of cell labelling parameters were investigated. The uptake of 57Co(II) oxine into erythrocytes was only 37%, and 65% of the activity eluted from the cells in cell-free plasma within 30 min. In contrast, high leukocyte and erythrocyte labelling efficiencies (> 90%) were obtained with 57Co(III) tropolonate containing cobalt carrier and the elution in cell-free plasma over 4 h was < 8%. High labelling efficiencies were also observed with 59Fe(III) oxine and 67Ga-MPO and the elution from leukocytes over 4 h was < 25%. We conclude that Co(III) tropolonate, Fe(III) oxine and Ga-MPO may be useful for radiolabelling leukocytes for PET investigations.

6-Alkoxymethyl-3-hydroxy-4H-pyranones: potential ligands for cell-labelling with indium.

We have identified ligands for cell labelling with indium-111: 3-hydroxy-6-propoxymethyl-4H-pyran-4-one and 6-butoxymethyl-3-hydroxy-4H-pyran-4-one. The leucocyte labelling efficiencies of (111)In complexes of these ligands were higher and label stabilities were found to be similar compared with those obtained using (111)In-tropolonate. High labelling efficiencies of neutrophils and lymphocytes were achieved with (111)In complexes of pyranones. Tropolone was found to have a greater inhibitory effect on metalloenzymes and to cause greater impairment of platelet function than 3-hydroxy-6-propoxymethyl-4H-pyran-4-one. Thus 6-alkoxymethyl-3-hydroxy-4H-pyran-4-ones may have advantages over current ligands used in cell labelling with (111)In.

Synthesis, physicochemical properties, and biological evaluation of hydroxypyranones and hydroxypyridinones: novel bidentate ligands for cell-labeling.

The synthesis of a range of hydroxypyranones and hydroxypyridinones with potential for the chelation of indium(III) is described. The crystal structures of two of the indium complexes are presented. The distribution coefficients of the ligands and the corresponding iron(III), gallium(III), and indium(III) complexes are reported. Good linear relationships between the distribution coefficients of the iron and gallium complexes and iron and indium complexes were obtained. In contrast a nonlinear relationship was obtained between the distribution coefficient of the free ligand and the distribution coefficient of the three groups of complexes. This latter relationship was used to identify compounds with optimal cell labeling properties. Two such compounds both 6-(alkoxymethyl)-3-hydroxy-4H-pyran-4-ones have been compared with tropolone for their ability to label human leucocytes with 111In. The leucocyte labeling efficiencies of the selected ligands were greater and the in-vitro plasma stabilities were similar to that of 111In-tropolonate. These results suggest that the new bidentate ligands may offer advantages over those currently used for cell-labeling.

Platelet labelling with indium-hydroxypyridinone and indium-hydroxypyranone complexes.

In order to identify new compounds which label platelets without affecting their function, three classes of metal chelating agents have been compared with oxine for their efficiency of indium-113m platelet labelling and for their short- and long-term effects on platelet function. The 3-hydroxypyridinones (both 2-ones and 4-ones) and 3-hydroxypyranones are bidentate chelators of trivalent metal ions that are neutrally charged in the metal-complexed form and hence gain access to cells readily. The hydroxypyranone ethylmaltol has been compared with the 3-hydroxypyridin-4-one CP94 and to its structurally related lipophilic analogue CP25 as well as with the 3-hydroxypyridin-2-one, CP02. The platelet labelling efficiencies with these ligands were between 75% and 95% of that obtained with oxine, following a 12-min incubation in saline. The optimal concentration for the hydroxypyridin-2-ones and hydroxypyridin-4-ones was approximately 10 microM compared with 100 microM for the hydroxypyranone ethylmaltol and 60 microM for oxine. Oxine and tropolone were found to produce significant inhibition of platelet aggregation to collagen in short-term experiments (10 min) or in longer term (18 and 42 h) ex vivo platelet cultures respectively. By contrast, ethylmaltol had no such inhibitory effects at either time interval. The relatively hydrophilic hydroxypyridin-4-one CP94 showed no inhibitory effects on collagen-induced aggregation in short-term studies, unlike the more lipid-soluble derivative CP25. These results suggest that ethylmaltol and related pyranones may have advantages over oxine and tropolone as indium platelet labelling agents where it is important not to damage platelets by the labelling process itself.

Urinary metabolic profiles in human and rat of 1,2-dimethyl- and 1,2-diethyl-substituted 3-hydroxypyridin-4-ones.

The urinary metabolic profiles of two novel orally active iron chelators, 1,2-dimethyl-3-hydroxypyridin-4-one (CP20 or L1) and 1,2-diethyl-3-hydroxypyridin-4-one (CP94), have been studied in rats. The metabolism of CP20 was also studied in humans. Four novel metabolites of CP20, and a further three metabolites of CP94 were characterized. CP20 was found to undergo extensive phase II metabolism at the 3-hydroxy position, forming predominantly the O-glucuronide, which accounted for 44% of the dose administered in rat and greater than 85% of the dose administered in man. The 3-O-methylated CP20 metabolite (metabolite I) accounted for 1% of the administered dose in both species, whereas the unmetabolized CP20 amounted to 10.5% and 4% of the dose administered in the rats and man, respectively. In contrast, CP94 was extensively hydroxylated at the 2-ethyl position to give its 2-(1-hydroxyethyl) metabolite in the rat, which accounted for 40% of the administered dose. The O-glucuronide metabolite of CP94 accounted for 13.8% of the administered dose, whereas the unmetabolized CP94 amounted to 6.9% of the administered dose. At 72 hr, urinary levels of CP20 and CP94 and their metabolites in the rat accounted for about 55-60% of the administered dose. A large portion of the dose is therefore probably eliminated via the bile. The identity of the above metabolites was established using a combination of two or more of the following techniques: fast atom bombardment-mass spectroscopy, LC-MS, UV-VIS spectroscopy, NMR spectroscopy, specific enzyme hydrolysis assays, and chemical synthesis of compounds.