Self-neutralizing oligonucleotides with enhanced cellular uptake.

There is tremendous potential for oligonucleotide (ON) therapeutics, but low cellular penetration due to their polyanionic nature is a major obstacle. We addressed this problem by developing a new approach for ON charge neutralization in which multiple branched charge-neutralizing sleeves (BCNSs) are attached to the internucleoside phosphates of ON by phosphotriester bonds. The BCNSs are terminated with positively charged amino groups, and are optimized to form ion pairs with the neighboring phosphate groups. The new modified ONs can be prepared by standard automated phosphoramidite chemistry in good yield and purity. They possess good solubility and hybridization properties, are not involved in non-standard intramolecular aggregation, have low cytotoxicity, adequate chemical stability, improved serum stability, and above all, display significantly enhanced cellular uptake. Thus, the new ON derivatives exhibit properties that make them promising candidates for the development of novel therapeutics or research tools for modulation of the expression of target genes.

Semi-Automated method of analysis of horizontal histological sections of skin for objective evaluation of fractional devices.

BACKGROUND AND OBJECTIVE: The treatment of skin with fractional devices creates columns of micro-ablation or micro-denaturation depending on the device. Since the geometric profiles of thermal damage depend on the treatment parameters or physical properties of the treated tissue, the size of these columns may vary from a few microns to a few millimeters. For objective evaluation of the damage profiles generated by fractional devices, this report describes an innovative and efficient method of processing and evaluating horizontal sections of skin using a novel software program. MATERIALS AND METHODS: Ex vivo porcine skin was treated with the Lux1540/10, Lux1540 Zoom and Lux2940 with 500 optics. Horizontal (radial) sections of biopsies were obtained and processed with H&E and NBTC staining. Digital images of the histologic sections were taken in either transmission or reflection illumination and were processed using the SAFHIR program. RESULTS: NBTC- and H&E-stained horizontal sections of ex vivo skin treated with ablative and non-ablative fractional devices were obtained. Geometric parameters, such as depth, diameter, and width of the coagulated layer (if applicable), and micro-columns of thermal damage, were evaluated using the SAFHIR software. The feasibility of objective comparison of the performance of two different fractional devices was demonstrated. CONCLUSION: The proposed methodology provides a comprehensive, objective, and efficient approach for the comparison of various fractional devices. Correlation of device settings with the objective dimensions of post-treatment damage profiles serve as a powerful tool for the prediction and modulation of clinical response.

Reversal of cystic fibrosis phenotype in a cultured Delta508 cystic fibrosis transmembrane conductance regulator cell line by oligonucleotide insertion.

Cystic fibrosis (CF) is a lethal genetic disorder that is due to mutations in the gene encoding the cAMP-activated anion CF transmembrane conductance regulator (CFTR) channel. A three-nucleotide base deletion (TTT), encoding phenylalanine in position 508 of the translatable CFTR sequence (accompanied by a C to T replacement immediately 5' to the deletion), accounts for approximately 75% of cases of the disease. In the present study, an oligonucleotide complex (CF4-CF6, 2'-0-methyl RNA-unmodified RNA oligonucleotide duplex, respectively) was used to restore CFTR function by insertion of missing bases in Delta508 CFTR mRNA from a cultured (Delta508) cell line. cAMP-activated whole-cell currents and Cl- transport were detected in CF4-CF6-treated, but not control Delta508, cells by patch-clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence (SPQ) quenching analyses, respectively. Further, the nucleotide addition in the deleted region of Delta508 CFTR was determined after amplification by RT-PCR. Insertion of UGU and replacement of U by C immediately 5' to the deletion site in Delta508 mRNA appear to have taken place, with phenotypic but not genotypic reversion in tissue culture of treated cells. The mechanism of insertion of nucleotides has yet to be determined.