Mustafa M. Munye

Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm

Background Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families. Methods and results Data from whole-exome sequencing in a consanguineous Turkish family, and whole-genome sequencing in the obligate carrier parents of a consanguineous Pakistani family was combined to identify homozygous loss-of-function mutations in ARMC4, segregating in all five affected individuals from both families. Both families carried nonsense mutations within the highly conserved armadillo repeat region of ARMC4: c.2675C>A; pSer892* and c.1972G>T; p.Glu658*. A deficiency of ARMC4 protein was seen in patients respiratory cilia accompanied by loss of the distal outer dynein arm motors responsible for generating ciliary beating, giving rise to cilia immotility. ARMC4 gene expression is upregulated during ciliogenesis, and we found a predicted interaction with the outer dynein arm protein DNAI2, mutations in which also cause PCD. Conclusions We report the first use of whole-genome sequencing to identify gene mutations causing PCD. Loss-of-function mutations in ARMC4 cause PCD with situs inversus and cilia immotility, associated with a loss of the distal outer (but not inner) dynein arms. This addition of ARMC4 to the list of genes associated with ciliary outer dynein arm defects expands our understanding of the complexities of PCD genetics.

The Role of the Helper Lipid on the DNA Transfection Efficiency of Lipopolyplex Formulations

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes.

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

PEGylation improves the receptor-mediated transfection efficiency of peptide-targeted, self-assembling, anionic nanocomplexes

Non-viral vector formulations comprise typically complexes of nucleic acids with cationic polymers or lipids. However, for in vivo applications cationic formulations suffer from problems of poor tissue penetration, non-specific binding to cells, interaction with serum proteins and cell adhesion molecules and can lead to inflammatory responses. Anionic formulations may provide a solution to these problems but they have not been developed to the same extent as cationic formulations due to difficulties of nucleic acid packaging and poor transfection efficiency. We have developed novel PEGylated, anionic nanocomplexes containing cationic targeting peptides that act as a bridge between PEGylated anionic liposomes and plasmid DNA. At optimized ratios, the components self-assemble into anionic nanocomplexes with a high packaging efficiency of plasmid DNA. Anionic PEGylated nanocomplexes were resistant to aggregation in serum and transfected cells with a far higher degree of receptor-targeted specificity than their homologous non-PEGylated anionic and cationic counterparts. Gadolinium-labeled, anionic nanoparticles, administered directly to the brain by convection-enhanced delivery displayed improved tissue penetration and dispersal as well as more widespread cellular transfection than cationic formulations. Anionic PEGylated nanocomplexes have widespread potential for in vivo gene therapy due to their targeted transfection efficiency and ability to penetrate tissues.

Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer

Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5–10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2–4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials.

Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector

Lipopolyplexes are of widespread interest for gene therapy due to their multifunctionality and high transfection efficiencies. Here we compared the biological and biophysical properties of a lipopolyplex formulation with its lipoplex and polyplex equivalents to assess the role of the lipid and peptide components in the formation and function of the lipopolyplex formulation. We show that peptide efficiently packaged plasmid DNA forming spherical, highly cationic nanocomplexes that are taken up efficiently by cells. However, transgene expression was poor, most likely due to endosomal degradation since the polyplex lacks membrane trafficking properties. In addition the strong peptide-DNA interaction may prevent plasmid release from the complex and so limit plasmid DNA availability. Lipid/DNA lipoplexes, on the other hand, produced aggregated masses that showed poorer cellular uptake than the polyplex but contrastingly greater levels of transgene expression. This may be due to the greater ability of lipoplexes relative to polyplexes to promote endosomal escape. Lipopolyplex formulations formed spherical, cationic nanocomplexes with efficient cellular uptake and significantly enhanced transfection efficiency. The lipopolyplexes combined the optimal features of lipoplexes and polyplexes showing optimal cell uptake, endosomal escape and availability of plasmid for transcription, thus explaining the synergistic increase in transfection efficiency.

Improved intracellular delivery of peptide- and lipid-nanoplexes by natural glycosides

Targeted nanocarriers undergo endocytosis upon binding to their membrane receptors and are transported into cellular compartments such as late endosomes and lysosomes. In gene delivery the genetic material has to escape from the cellular compartments into the cytosol. The process of endosomal escape is one of the most critical steps for successful gene delivery. For this reason synthetic lipids with fusogenic properties such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are integrated into the nanocarriers. In this study we show that a natural, plant derived glycoside (SO1861) from Saponaria officinalis L. greatly improves the efficacy of lipid based as well as non-lipid based targeted nanoplexes consisting of a targeted K16 peptide with a nucleic acid binding domain and plasmid-DNA, minicircle-DNA or small interfering RNA (siRNA). By confocal live cell imaging and single cell analyses, we demonstrate that SO1861 augments the escape of the genetic cargo out of the intracellular compartments into the cytosol. Co-localisation experiments with fluorescence labelled dextran and transferrin indicate that SO1861 induces the release of the genetic cargo out of endosomes and lysosomes. However, the transduction efficacy of a lentivirus based gene delivery system was not augmented. In order to design receptor-targeted nanoplexes (LPD) with improved functional properties, SO1861 was integrated into the lipid matrix of the LPD. The SO1861 sensitized LPD (LPDS) were characterized by dynamic light scattering and transmission electron microscopy. Compared to their LPD counterparts the LPDS-nanoplexes showed a greatly improved gene delivery. As shown by differential scanning calorimetry SO1861 can be easily integrated into the lipid bilayer of glycerophospholipid model membranes. This underlines the great potential of SO1861 as a new transfection multiplier for non-viral gene delivery systems.

COLEC10 is mutated in 3MC patients and regulates early craniofacial development

3MC syndrome is an autosomal recessive heterogeneous disorder with features linked to developmental abnormalities. The main features include facial dysmorphism, craniosynostosis and cleft lip/palate; skeletal structures derived from cranial neural crest cells (cNCC). We previously reported that lectin complement pathway genes COLEC11 and MASP1/3 are mutated in 3MC syndrome patients. Here we define a new gene, COLEC10, also mutated in 3MC families and present novel mutations in COLEC11 and MASP1/3 genes in a further five families. The protein products of COLEC11 and COLEC10, CL-K1 and CL-L1 respectively, form heteromeric complexes. We show COLEC10 is expressed in the base membrane of the palate during murine embryo development. We demonstrate how mutations in COLEC10 (c.25C>T; p.Arg9Ter, c.226delA; p.Gly77Glufs*66 and c.528C>G p.Cys176Trp) impair the expression and/or secretion of CL-L1 highlighting their pathogenicity. Together, these findings provide further evidence linking the lectin complement pathway and complement factors COLEC11 and COLEC10 to morphogenesis of craniofacial structures and 3MC etiology.

Delivery of ENaC siRNA to epithelial cells mediated by a targeted nanocomplex: a therapeutic strategy for cystic fibrosis

The inhibition of ENaC may have therapeutic potential in CF airways by reducing sodium hyperabsorption, restoring lung epithelial surface fluid levels, airway hydration and mucociliary function. The challenge has been to deliver siRNA to the lung with sufficient efficacy for a sustained therapeutic effect. We have developed a self-assembling nanocomplex formulation for siRNA delivery to the airways that consists of a liposome (DOTMA/DOPE; L), an epithelial targeting peptide (P) and siRNA (R). LPR formulations were assessed for their ability to silence expression of the transcript of the gene encoding the α-subunit of the sodium channel ENaC in cell lines and primary epithelial cells, in submerged cultures or grown in air-liquid interface conditions. LPRs, containing 50 nM or 100 nM siRNA, showed high levels of silencing, particularly in primary airway epithelial cells. When nebulised these nanocomplexes still retained their biophysical properties and transfection efficiencies. The silencing ability was determined at protein level by confocal microscopy and western blotting. In vivo data demonstrated that these nanoparticles had the ability to silence expression of the α-ENaC subunit gene. In conclusion, these findings show that LPRs can modulate the activity of ENaC and this approach might be promising as co-adjuvant therapy for cystic fibrosis.

A method for concentrating lipid peptide DNA and siRNA nanocomplexes that retains their structure and transfection efficiency

Nonviral gene and small interfering RNA (siRNA) delivery formulations are extensively used for biological and therapeutic research in cell culture experiments, but less so in in vivo and clinical research. Difficulties with formulating the nanoparticles for uniformity and stability at concentrations required for in vivo and clinical use are limiting their progression in these areas. Here, we report a simple but effective method of formulating monodisperse nanocomplexes from a ternary formulation of lipids, targeting peptides, and nucleic acids at a low starting concentration of 0.2 mg/mL of DNA, and we then increase their concentration up to 4.5 mg/mL by reverse dialysis against a concentrated polymer solution at room temperature. The nanocomplexes did not aggregate and they had maintained their biophysical properties, but, importantly, they also mediated DNA transfection and siRNA silencing in cultured cells. Moreover, concentrated anionic nanocomplexes administered by convection-enhanced delivery in the striatum showed efficient silencing of the β-secretase gene BACE1. This method of preparing nanocomplexes could probably be used to concentrate other nonviral formulations and may enable more widespread use of nanoparticles in vivo.