The Skin You Are In: Design-of-Experiments Optimization of Lipid Nanoparticle Self-Amplifying RNA Formulations in Human Skin Explants.

Messenger RNA (mRNA) is a promising tool for biotherapeutics, and self-amplifying mRNA (saRNA) is particularly advantageous, because it results in abundant protein expression and production is easily scalable. While mRNA therapeutics have been shown to be highly effective in small animals, the outcomes do not scale linearly when these formulations are translated to dose-escalation studies in humans. Here, we utilize a design of experiments (DoE) approach to optimize the formulation of saRNA lipid nanoparticles in human skin explants. We first observed that luciferase expression from saRNA peaked after 11 days in human skin. Using DoE inputs of complexing lipid identity, lipid nanoparticle dose, lipid concentration, particle concentration, and ratio of zwitterionic to cationic lipids, we optimized the saRNA-induced luciferase expression in skin explants. Lipid identity and lipid concentration were found to be significant parameters in the DoE model, and the optimized formulation resulted in ∼7-fold increase in luciferase expression, relative to initial 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) formulation. Using flow cytometry, we observed that optimized formulations delivered the saRNA to ∼2% of the resident cells in the human skin explants. Although immune cells comprise only 7% of the total population of cells in skin, immune cells were found to express ∼50% of the RNA. This study demonstrates the powerful combination of using a DoE approach paired with clinically relevant human skin explants to optimize nucleic acid formulations. We expect that this system will be useful for optimizing both formulation and molecular designs of clinically translational nucleic acid vaccines and therapeutics.

A method to select patients for vertical scar or inverted-T pattern breast reduction.

Vertical scar breast reduction (VSBR) is an increasingly popular technique but can carry a high complication and revision rate. We evaluate our complication rate when selecting patients for VSBR or inverted-T breast reductions (ITBR). We looked at 133 consecutive primary bilateral breast reductions performed by a single consultant from 1998 to 2003 (23 incomplete records). VSBR (82 breasts) and ITBR (120 breasts) were performed, with glandular or superomedial pedicle transposition of the nipple-areola complex (NAC), or free nipple grafts (FNG) (18 breasts). The pattern of skin excision chosen was determined by the degree of skin shrinking required to achieve the correct vertical height of the new breast. If the measurement from the inframammary fold to the lower border of the new areola position was < 18 cm the patient underwent VSBR. We assessed complication and revision rates in each group. The complication rate (haematoma, infection, delayed healing, nipple/fat necrosis) in VSBR was 11%, in ITBR 23% (P<0.05). Revision rate was 2.4% in the VSBR and 3.3% in ITBR group. The VSBR had no problem scars, while the ITBR produced problem scars in 16% of operated breasts (P<0.001). We feel it is possible to reduce skin to 40-50% of its original length. Attempting a greater degree of shrinkage produces a high rate of revision surgery. We suggest a method of selection of technique by a reproducible measurement of the vertical skin reduction to produce a low complication rate and low revision rate.