The hair follicle bulge possesses putative epithelial stem cells. Characterization of these cells has been hampered by the inability to target bulge cells genetically. Here, we use a Keratin1-15 (Krt1-15, also known as K15) promoter to target mouse bulge cells with an inducible Cre recombinase construct or with the gene encoding enhanced green fluorescent protein (EGFP), which allow for lineage analysis and for isolation of the cells. We show that bulge cells in adult mice generate all epithelial cell types within the intact follicle and hair during normal hair follicle cycling. After isolation, adult Krt1-15-EGFP-positive cells reconstituted all components of the cutaneous epithelium and had a higher proliferative potential than Krt1-15-EGFP-negative cells. Genetic profiling of hair follicle stem cells revealed several known and unknown receptors and signaling pathways important for maintaining the stem cell phenotype. Ultimately, these findings provide potential targets for the treatment of hair loss and other disorders of skin and hair.

Viral gene therapy has high efficacy, but is plagued by serious safety risks, production and manufacturing challenges, and other limitations including nucleic acid cargo capacity. [1] In contrast, non-viral gene delivery systems, while addressing these challenges, remain less effective.[2] Here we develop end-modified poly(b-amino ester)s, easy-to-synthesize degradable polymers, that are able to deliver DNA to primary human umbilical vein endothelial cells (HUVECs) at levels comparable to adenovirus at a Multiplicity of Infection (MOI) between 100 and 500, and two orders of magnitude better than the commonly used non-viral polymeric vector, polyethylenimine (PEI). Interestingly, small structural changes were found to have dramatic effects on multiple steps of gene delivery including the DNA binding affinity, nanoparticle size, intracellular DNA uptake, and final protein expression. In vivo, these polymer modifications dramatically enhance DNA delivery to ovarian tumors. We believe the development of polymeric vectors with gene delivery efficacy comparable to adenovirus could set a new benchmark in nonviral transfection capability. Numerous polymeric materials have been used for gene delivery including poly(L-lysine), polyethylenimine, poly(amidoamine) dendrimers, poly(a-[4-aminobutyl]-L-glycolic acid), chitosan, cyclodextrin, and others. [2,3] While significant strides have been made in improving delivery, efficacy remains generally low, particularly for primary cells in the presence of serum.[4] Poly(b-amino ester) s are promising materials that bind and self-assemble with DNA to form stable nanoparticles that effectively enter cells, escape the endosomal compartments, and degrade via hydrolytic cleavage of backbone ester groups. [5][6][7][8] While good in vitro and in vivo activity has been described, [5,9] structural diversity of existing poly(b-amino ester) was limited by chemical requirements of conjugate addition. [5][6][7][8]10] We hypothesized that the exploration of an expanded chemical space through combinatorial modification of poly(b-amino ester) s could optimize performance. To this end, and to better understand structure-function relationships, we synthesized a library of end-modified poly(b-amino ester) s using three base diacrylate-terminated polymers and twelve amine end-capping reagents (Fig. 1). Chemical methods were developed to allow a simple, one step modification of base polymers with several different amine capping agents (see methods). In this way, the combined effects of internal structure and amine termination on poly(b-amino ester) transfections could be systematically assessed. Once synthesized, polymers were characterized by 1 H NMR and GPC (see methods).Three different polymers were chosen for end modification: C32, D60 and C20 (Fig. 1B). High-throughput screening studies have identified polymer C32 as the most effective poly(bamino ester) to date for gene delivery. [5,6] Another polymer, D60 is also an effective gene delivery agent, but with a structure significantly different from...

. To this end, we generated a library of >500 degradable, poly(␤-amino esters) for potential use as nonviral DNA vectors. Using high-throughput methods, we screened this library in vitro for transfection efficiency and cytotoxicity. We tested the best performing polymer, C32, in mice for toxicity and DNA delivery after intratumor and i.m. injection. C32 delivered DNA intratumorally Ϸ4-fold better than one of the best commercially available reagents, jetPEI (polyethyleneimine), and 26-fold better than naked DNA. Conversely, the highest transfection levels after i.m. administration were achieved with naked DNA, followed by polyethyleneimine; transfection was rarely observed with C32. Additionally, polyethyleneimine induced significant local toxicity after i.m. injection, whereas C32 demonstrated no toxicity. Finally, we used C32 to deliver a DNA construct encoding the A chain of diphtheria toxin (DT-A) to xenografts derived from LNCaP human prostate cancer cells. This construct regulates toxin expression both at the transcriptional level by the use of a chimeric-modified enhancer͞promoter sequence of the human prostate-specific antigen gene and by DNA recombination mediated by Flp recombinase. C32 delivery of the A chain of diphtheria toxin DNA to LNCaP xenografts suppressed tumor growth and even caused 40% of tumors to regress in size. Because C32 transfects tumors locally at high levels, transfects healthy muscle poorly, and displays no toxicity, it may provide a vehicle for the local treatment of cancer.prostate ͉ cationic polymers