Mutations in Three Genes Encoding Proteins Involved in Hair Shaft Formation Cause Uncombable Hair Syndrome.

Uncombable hair syndrome (UHS), also known as "spun glass hair syndrome," "pili trianguli et canaliculi," or "cheveux incoiffables" is a rare anomaly of the hair shaft that occurs in children and improves with age. UHS is characterized by dry, frizzy, spangly, and often fair hair that is resistant to being combed flat. Until now, both simplex and familial UHS-affected case subjects with autosomal-dominant as well as -recessive inheritance have been reported. However, none of these case subjects were linked to a molecular genetic cause. Here, we report the identification of UHS-causative mutations located in the three genes PADI3 (peptidylarginine deiminase 3), TGM3 (transglutaminase 3), and TCHH (trichohyalin) in a total of 11 children. All of these individuals carry homozygous or compound heterozygous mutations in one of these three genes, indicating an autosomal-recessive inheritance pattern in the majority of UHS case subjects. The two enzymes PADI3 and TGM3, responsible for posttranslational protein modifications, and their target structural protein TCHH are all involved in hair shaft formation. Elucidation of the molecular outcomes of the disease-causing mutations by cell culture experiments and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild-type proteins. Scanning electron microscopy observations revealed morphological alterations in hair coat of Padi3 knockout mice. All together, these findings elucidate the molecular genetic causes of UHS and shed light on its pathophysiology and hair physiology in general.

Recent Progress of Polymeric Nanogels for Gene Delivery.

With its nearly unrestricted possibilities, gene therapy attracts more and more significance in modern-day research. The only issue still seeming to hold back its clinical success is the actual effective delivery of genetic material. Nucleic acids are in general challenging to administer to their intracellular targets due to their unfavorable pharmaceutical characteristics. Polymeric nanogels present a promising delivery platform for oligonucleotide-based therapies, as the growing number of reports deliberated in this review represents. Within the scope of this article, recent progress in the employment of nanogels as gene delivery vectors is summarized and different examples of modified, stimuli-responsive, targeted and co-delivering nanogels are discussed in detail. Furthermore, major aspects of successful gene delivery are addressed and critically debated in regards to nanogels, giving insights into what progress has been made and which key issues still need to be further approached.

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.

A Method for Targeted Nonviral siRNA Delivery in Cancer and Inflammatory Diseases.

Small interfering RNA (siRNA)-based therapy has been subject of intense research since the discovery of RNA interference (RNAi), providing a tool to potentially silence any chosen gene. Nevertheless, efficient delivery still presents a major hurdle to translating this promising technology into medical practice. Here, we describe a straightforward method to prepare and characterize an effective delivery system consisting of low-molecular-weight polyethylenimine (PEI) and transferrin (Tf). Tf-PEI polyplexes are not only able to successfully transport and protect the sensitive nucleic acid payload from degradation but also to selectively deliver the siRNA to transferrin receptor (TfR)-overexpressing cells, playing key roles in the pathology of numerous cancer types as well as inflammatory diseases.

Pulmonary delivery of siRNA as a novel treatment for lung diseases.

Graphical abstract.

Evaluating the Regulation of Cytokine Levels After siRNA Treatment in Antigen-Specific Target Cell Populations via Intracellular Staining.

RNA interference (RNAi) offers a promising base for therapeutic knockdown of clinically relevant genes. Local delivery routes as well as targeted delivery to specific cell populations have been shown to circumvent several hurdles of successful siRNA delivery in vivo. To evaluate and quantify the treatment effect in a precise way, next to measuring the downregulation on gene and protein levels, it is equally essential to investigate the influence on downstream factors such as generated cytokines. Here, we describe an expressive method to specifically isolate the desired target cells and determine their levels of intracellular cytokines by flow cytometry using the example of murine lungs after pulmonary in vivo transfection with siRNA.Therefore, the lungs of treated mice are harvested and processed into single cell suspensions, in which CD4 positive T cells are marked by antibody-coupled magnetic beads and isolated via magnetic separation. These purified target cells are then fixed and permeabilized, making their intracellular interleukins accessible for staining with fluorescently labeled antibodies. Thus, the cytokine levels and hence the precise influence of the siRNA treatment on intracellular conditions can be measured.

Coming in and Finding Out: Blending Receptor-Targeted Delivery and Efficient Endosomal Escape in a Novel Bio-Responsive siRNA Delivery System for Gene Knockdown in Pulmonary T Cells.

RNA interference (RNAi) offers the potential to selectively silence disease-related genes in defined cell subsets. Translation into the clinical routine is, however, still hampered by the lack of efficient carrier systems for therapeutic siRNA, endosomal entrapment presenting a major hurdle. A promising siRNA delivery system has previously been developed on the base of polyethylenimine (PEI) and the targeting ligand transferrin (Tf) to specifically reach activated T cells in the lung. In the present work, the focus is on optimizing Tf-PEI polyplexes for gene knockdown in primary activated T cells by improving their endosomal escape properties. Blending of the conjugate with membrane lytic melittin significantly enhanced endosomal release and thereby cytoplasmic delivery, while maintaining selective T cell targeting abilities and overall cell tolerability. The gathered data furthermore demonstrate that melittin addition also distinctly improves several other essential particle characteristics, such as siRNA encapsulation efficiency and stability in lung lining fluids. In conclusion, this results in a novel upgraded siRNA delivery system that is not only able to specifically deliver its payload to the desired target cells via receptor-mediated endocytosis, but also shows enhanced release from endosomal vesicles in order to initiate RNAi in the cytoplasm.

In vitro and in vivo delivery of siRNA via VIPER polymer system to lung cells.

The block copolymer VIPER (virus-inspired polymer for endosomal release) has been reported to be a promising novel delivery system of DNA plasmids both in vitro and in vivo. VIPER is comprised of a polycation segment for condensation of nucleic acids as well as a pH-sensitive segment that exposes the membrane lytic peptide melittin in acidic environments to facilitate endosomal escape. The objective of this study was to investigate VIPER/siRNA polyplex characteristics, and compare their in vitro and in vivo performance with commercially available transfection reagents and a control version of VIPER lacking melittin. VIPER/siRNA polyplexes were formulated and characterized at various charge ratios and shown to be efficiently internalized in cultured cells. Target mRNA knockdown was confirmed by both flow cytometry and qRT-PCR and the kinetics of knockdown was monitored by live cell spinning disk microscopy, revealing knockdown starting by 4 h post-delivery. Intratracheal instillation of VIPER particles formulated with sequence specific siRNA to the lung of mice resulted in a significantly more efficient knockdown of GAPDH compared to treatment with VIPER particles formulated with scrambled sequence siRNA. We also demonstrated using pH-sensitive labels that VIPER particles experience less acidic environments compared to control polyplexes. In summary, VIPER/siRNA polyplexes efficiently deliver siRNA in vivo resulting in robust gene silencing (>75% knockdown) within the lung.

Biodegradable Three-Layered Micelles and Injectable Hydrogels.

Polymeric micelles have found a growing interest as gene vectors due to the serious safety concerns associated with viral vectors. In particular, the cationic polymer polyethylene imine (PEI) has shown relatively high condensation and transfection efficiencies. Additionally, polyethylene glycol (PEG) modification of polymeric gene vectors has dramatically improved their biological properties, including enhanced biocompatibility, prolonged circulation time, and increased bio-distribution. However, PEG grafting of PEI for subsequent condensation of nucleic acids (NAs) does not necessarily result in the formation of a PEI/NAs core with a PEG corona. But often times, the presence of PEG interferes with PEI's electrostatic interaction with NAs. We describe here a facile method to prepare multilayered biodegradable micelles which address some of the critical drawbacks associated with current PEI-based systems. The polyplex micelles have superb stability and stealth properties. Moreover, we describe a method to prepare fully biodegradable and biocompatible injectable hydrogels for use in localized gene therapy.

Folate receptor targeted three-layered micelles and hydrogels for gene delivery to activated macrophages.

New folic acid (FA) coupled three layered micelles (3LM) were designed to encapsulate DNA, and their application as delivery system that specifically targets activated macrophages was investigated for new treatment options in rheumatoid arthritis (RA). FA coupled poly(l-lactide)-b-poly(ethylene glycol) (FA-PEG-PLLA) was synthesized via the NHS-ester activated/amine coupling method. Fluorescein labeled folic acid was used for flow cytometric detection of the expression of functional folic receptor ß in LPS-activated and resting macrophages. FA coupled 3LM were formulated in a two-step procedure and characterized regarding hydrodynamic diameters and zeta potentials. The presence of the targeting ligand was shown not to increase the size of the 3LM compared to their non-targeted counterparts. Targeted and non-targeted 3LM were used in vitro to optimize uptake conditions in the RAW 264.7 macrophage cell line. The amount of FA coupled polymer in the final formulation was found to be optimal at 75% FA-PEG-PLLA and 25% PLLA-PEG-PLLA. Subsequently, transgene expression in vitro in RAW 264.7 cells and ex vivo in primary activated and resting mouse macrophages was determined as a function of FR-mediated internalization of 3LM encapsulating GFP expressing plasmid. FR-overexpressing activated cells, as successfully identified by internalization of FA-fluorescein, showed significantly higher GFP expression in vitro and ex vivo than resting macrophages with only a basal level of FR expression. Lastly, injectable hydrogels as depot formulation were formed by stereocomplexation, and their degradation, DNA release profiles, and dissociation into intact 3LM were found to be beneficial for potential in vivo application. Our findings confirm that FA-3LM are taken up by activated macrophages via folate receptor mediated endocytosis and that their hydrogels release intact 3LM for efficient transfection of primary macrophages. Therefore, FA-3LM could become a promising delivery system for receptor-mediated drug or gene delivery and novel therapy for rheumatoid arthritis in an in situ forming gel formulation.

Three-Layered Biodegradable Micelles Prepared by Two-Step Self-Assembly of PLA-PEI-PLA and PLA-PEG-PLA Triblock Copolymers as Efficient Gene Delivery System.

"Three-layered micelles" (3LM) composed of two triblock copolymers, poly(L-lactide)-b-polyethyleneimine-b-poly(L-lactide) (PLLA-PEI-PLLA) and poly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide) (PLLA-PEG-PLLA) are designed to combine electrostatic interaction and solvent-induced condensation of DNA. The low molecular weight PLLA-PEI-PLLA is synthesized by a facile amine-protection/deprotection approach and employed as a gene vector, compacting DNA as a polyplex core in the organo-micelles. The individual organo-micelle is further encapsulated within a PLLA-PEG-PLLA amphiphilic micelle leading to an aqueous stable colloidal dispersion. The resulting spherical 3LM possess a hydrodynamic diameter of ca. 200 nm and zeta potential close to neutral and display excellent stability to competing polyanions such as dextran sulfate in neutral pH (7.4). Such high stability is attributed to the complete shielding of the PEI/DNA polyplex core with an impermeable hydrophobic intermediate layer. However, greater than 90% of the encapsulated DNA are released within 30 min when exposed to slightly acidic pH (4.5). Based on our findings, a new class of non-viral delivery system for nucleic acids with superb stability and stealth properties is identified.