Identification of the minimum peptide from mouse myostatin prodomain for human myostatin inhibition.

Myostatin, an endogenous negative regulator of skeletal muscle mass, is a therapeutic target for muscle atrophic disorders. Here, we identified minimum peptides 2 and 7 to effectively inhibit myostatin activity, which consist of 24 and 23 amino acids, respectively, derived from mouse myostatin prodomain. These peptides, which had the propensity to form α-helix structure, interacted to myostatin with KD values of 30-36 nM. Moreover, peptide 2 significantly increased muscle mass in Duchenne muscular dystrophy model mice.

Dynamic amphiphile libraries to screen for the "fragrant" delivery of siRNA into HeLa cells and human primary fibroblasts.

Dynamic amphiphiles are amphiphiles with dynamic covalent bridges between their hydrophilic heads and their hydrophobic tails. Their usefulness to activate ion transporters, for odorant release, and for differential sensing of odorants and perfumes, has been demonstrated recently. Here, we report that the same "fragrant" dynamic amphiphiles are ideal to screen for new siRNA transfection agents. The advantages of this approach include rapid access to fairly large libraries of complex structures, and possible transformation en route to assist uptake and minimize toxicity. We report single-component systems that exceed the best commercially available multicomponent cocktails with regard to both efficiency and velocity of EGFP knockdown in HeLa cells. In human primary fibroblasts, siRNA-mediated enzyme knockdown nearly doubled from >30% for Lipofectamine to >60% for our best hit. The identified structures were predictable neither from literature nor from results in fluorogenic vesicles and thus support the importance of conceptually innovative screening approaches.

KALA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine.

DNA vaccines are a new-generation vaccines that elicit an immunological response against a wide-variety of antigens with frequent mutations. However, an effective non-viral vector for genetically engineered DNA to dendritic cells is yet to be developed. We previously reported that an octaarginine (R8)-modified tetra-lamellar multi-functional envelope-type nano device (R8-T-MEND) increases transfection efficiency in dendritic cell cultures (JAWS II). The critical structural elements of the R8-T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes, and two endosome-fusogenic outer envelopes. While the gene expression was drastically enhanced by R8-T-MEND, antigen presentation using an epitope-encoding plasmid DNA remains an obstacle for future non-viral vectors in DNA vaccinations. In the present study, we upgraded the function of R8-T-MEND by improving the membrane-fusion processes with endosome- and nuclear membranes by incorporating the KALA peptide, and by reducing the charge ratio (+/-), in an attempt to accelerate intra-nuclear decondensation. The resulting KALA-modified T-MEND (R8/KALA-T-MEND) showed an approximately 20-fold higher transgene expression compared with the conventional R8-T-MEND in JAWS II, and exceeded that of Lipofectamine PLUS, a commercially available transfection reagent. Furthermore, significant antigen presentation of a specific epitope (SIINFEKL) was observed for the R8/KALA-T-MEND but was not detected for the conventional T-MEND or Lipofectamine PLUS when an ovalbumin (OVA)-encoding plasmid DNA was transfected. It thus appears that the R8/KALA-T-MEND has the potential for use as a vector in DNA vaccinations.