Delivering mRNAs to mouse tissues using the SEND system.

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo . Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum phosphofructokinase.

The nucleotide sequence of a full-length cDNA encoding phosphofructokinase (PFK) enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2,653 bases comprises a single open reading frame of 2,452 bases and a noncoding region of 201 bases after the stop codon. The mature protein contains 812 amino acids and has a molecular mass of 90,900 Da. The amino acid sequences of several peptides derived from the purified protein show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the protein with those of 3 other worms as well as those of human, rabbit, and bacterial enzymes reveals highly conserved regions interrupted with stretches of lesser sequence similarity. Analyses of the subunit primary structure reveal, as in other eukaryotic PFKs, that the amino-terminal half is homologous to the carboxy-terminal half, supporting the hypothesis that the PFK gene evolved by duplication of the prokaryotic gene and that the allosteric sites arose by mutations at the catalytic site. The location of the phosphorylation site is unique and different compared with other PFKs and plays a key role in regulation of the enzyme activity. Structural motifs such as the putative substrate and effector binding domains and also the key amino acids involved therein are clearly identified by alignment of all the PFK protein sequences.

Genotype-phenotype associations for common CYP3A4 and CYP3A5 variants in the basal and induced metabolism of midazolam in European- and African-American men and women.

CYP3A activity in adults varies between individuals and it has been suggested that this has a genetic basis, possibly related to variant alleles in CYP3A4 and CYP3A5 genes. Accordingly, genotype-phenotype associations were investigated under constitutive and induced conditions. Midazolam's systemic and oral clearances, and the erythromycin breath test (ERBT) were determined in 57 healthy subjects: 23 (11 men, 12 women) European- and 34 (14 men, 20 women) African-Americans. Studies were undertaken in the basal state and after 14-15 days pretreatment with rifampin. DNA was characterized for the common polymorphisms CYP3A4*1B, CYP3A5*3, CYP3A5*6 and CYP3A5*7 by direct sequencing, and for exon 21 and exon 26 variants of MDR1 by allele-specific, real-time polymerase chain reaction. In 95% of subjects, the basal systemic clearance of midazolam was unimodally distributed and variability was less than four-fold whereas, in 98% of the study population, oral clearance varied five-fold. No population or sex-related differences were apparent. Similar findings were observed with the ERBT. Rifampin pretreatment markedly increased the systemic (two-fold) and oral clearance (16-fold) of midazolam, and the ERBT (two-fold) but the variabilities were unchanged. No associations were noted between these phenotypic measures and any of the studied genotypes, except for oral clearance and its fold-increase after rifampin. These were related to the presence of CYP3A4*1B and the inversely linked CYP3A5*3 polymorphism, with the extent of induction being approximately 50% greater in CYP3A5*3 homozygotes compared to wild-type subjects. In most healthy subjects, variability in intestinal and hepatic CYP3A activity, using midazolam as an in-vivo probe, is modest and common polymorphisms in CYP3A4 and CYP3A5 do not appear to have important functional significance.