Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

Delta-aminolevulinic acid synthase 2 expression in combination with iron as modifiers of disease severity in erythropoietic protoporphyria.

Deficiency in ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway, leads to an accumulation of protoporphyrin IX (PPIX) that causes a severely painful phototoxic reaction of the skin in patients with erythropoietic protoporphyria (EPP). Besides phototoxicity of the skin, EPP patients often present with symptoms of iron deficiency in form of a microcytic and hypochromic anemia with low serum iron and ferritin. In addition, elevated aminolevulinic acid synthase 2 (ALAS2) both at the mRNA and protein levels have been observed among EPP patients. ALAS is the first enzyme in the pathway and exists in two isoforms, whereby the isoform 2 (ALAS2) is expressed exclusively in erythropoiesis. The mRNA of ALAS2 contains an iron response element (IRE) at its 5'UTR. When iron is limited, iron response element binding protein 2 (IRP2) binds to the IRE of ALAS2 mRNA and suppresses its translation. In this study, we demonstrated that iron deprivation increased the amount of ALAS2 mRNA as well as the ratio of ALAS2 to FECH mRNAs in cultured erythroleukemic K562 cells. At the protein level, however, iron deprivation in the cell line caused reductions in both enzymes as shown by the Western blot analysis. A comparable increase in the ratio of ALAS2 to FECH mRNAs was also found in EPP patients indicating an imbalance in heme biosynthesis. As iron cannot be completely missing from an organism, we assume that in EPP patients, a certain amount of ALAS2 mRNA is translated despite a partial deficiency of FECH. The increase in ALAS2 enzyme contributes to the accumulation in PPIX in the patients. Targeted inhibition of ALAS2 could therefore be a treatment option for EPP.

Autologous Cell Therapy Approach for Duchenne Muscular Dystrophy using PiggyBac Transposons and Mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease currently without cure. We investigated the use of the PiggyBac transposon for full-length dystrophin expression in murine mesoangioblast (MABs) progenitor cells. DMD murine MABs were transfected with transposable expression vectors for full-length dystrophin and transplanted intramuscularly or intra-arterially into mdx/SCID mice. Intra-arterial delivery indicated that the MABs could migrate to regenerating muscles to mediate dystrophin expression. Intramuscular transplantation yielded dystrophin expression in 11%-44% of myofibers in murine muscles, which remained stable for the assessed period of 5 months. The satellite cells isolated from transplanted muscles comprised a fraction of MAB-derived cells, indicating that the transfected MABs may colonize the satellite stem cell niche. Transposon integration site mapping by whole-genome sequencing indicated that 70% of the integrations were intergenic, while none was observed in an exon. Muscle resistance assessment by atomic force microscopy indicated that 80% of fibers showed elasticity properties restored to those of wild-type muscles. As measured in vivo, transplanted muscles became more resistant to fatigue. This study thus provides a proof-of-principle that PiggyBac transposon vectors may mediate full-length dystrophin expression as well as functional amelioration of the dystrophic muscles within a potential autologous cell-based therapeutic approach of DMD.

Characterization of the algC gene expression pattern in the multidrug resistant Acinetobacter baumannii AIIMS 7 and correlation with biofilm development on abiotic surface.

Relative quantification of algC gene expression was evaluated in the multidrug resistant strain Acinetobacter baumannii AIIMS 7 biofilm (3 to 96 h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05). Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h). Cloning, heterologous expression, and bioinformatics analyses indicated algC gene product as the bifunctional enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) of ∼ 53 kDa size, which augmented biofilms significantly in algC clones compared to controls (lacking algC gene), further localized by scanning electron microscopy. Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances). Our observation on differential expression pattern of algC having strong correlation with important biofilm stages, scanning electron-microscopic evidence of biofilm augmentation taken together with predictive enzyme functions via molecular dynamic (MD) simulation, proposes a new basis of A. baumannii AIIMS 7 biofilm development on inanimate surfaces.

Characterization of eDNA from the clinical strain Acinetobacter baumannii AIIMS 7 and its role in biofilm formation.

Release of extracellular DNA (eDNA) was observed during in vitro growth of a clinical strain of Acinetobacter baumannii. Membrane vesicles (MV) of varying diameter (20-200 nm) containing DNA were found to be released by transmission electron microscopy (TEM) and atomic force microscopy (AFM). An assessment of the characteristics of the eDNA with respect to size, digestion pattern by DNase I/restriction enzymes, and PCR-sequencing, indicates a high similarity with genomic DNA. Role of eDNA in static biofilm formed on polystyrene surface was evaluated by biofilm augmentation assay using eDNA available in different preparations, for example, whole cell lysate, cell-free supernatant, MV suspension, and purified eDNA. Biofilm augmentation was seen up to 224.64%, whereas biofilm inhibition was 59.41% after DNase I treatment: confirming that eDNA facilitates biofilm formation in A. baumannii. This is the first paper elucidating the characteristics and role of eDNA in A. baumannii biofilm, which may provide new insights into its pathogenesis.

Rheological properties of skeletal muscles in a Duchenne muscular dystrophy murine model before and after autologous cell therapy.

Duchenne muscular dystrophy (DMD) is still an incurable muscle degenerative disease; thus, numerous studies focused on novel therapeutic approaches. However, a simple assay of muscle function restoration remains needed. Herein, we used an oscillatory shear rheometer to evaluate changes in rheological properties of mouse muscles (tibialis anterior, TA) and their restoration upon autologous cell therapy by comparing the viscoelastic properties of normal, diseased and treated muscles. Amplitude sweep tests of muscle samples were performed under 20% compression over a range of shear strain between 0.01 and 2% and frequency of 1 rad/s. The samples were tested in plane-plane geometry and horizontal myofiber alignment. Typical linear viscoelastic region (LVER) patterns were found for each muscle type. For healthy muscles, a broad LVER between shear deformations (γ) of 0.013-0.62% was observed. The LVER of DMD mdx/SCID muscles was found at 0.14% to 0.46% shear deformation, and no shear dependence of storage (G') and loss (G") moduli at γ range changing from 0.034% to 0.26% was found for transplanted tissues. G'LVER and G"LVER moduli of healthy muscles were significantly higher than G'LVER and G"LVER of dystrophic tissues. Additionally, muscle resistance assessment by rheometer indicated that muscles transplanted with stem cells restored elastic properties to levels close to those of healthy muscles. Interestingly, histological staining and rheological data indicate that the loss factor is strongly related to structural changes of examined muscles.

Repurposing of glycine transport inhibitors for the treatment of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare disease in which patients experience severe light sensitivity. It is caused by a deficiency of ferrochelatase (FECH), the last enzyme in heme biosynthesis (HBS). The lack of FECH causes accumulation of its photoreactive substrate protoporphyrin IX (PPIX) in patients' erythrocytes. Here, we explored an approach for the treatment of EPP by decreasing PPIX synthesis using small-molecule inhibitors directed to factors in the HBS pathway. We generated a FECH-knockout clone from K562 erythroleukemia cells, which accumulates PPIX and undergoes oxidative stress upon light exposure. We used these matched cell lines to screen a set of publicly available inhibitors of factors in the HBS pathway. Inhibitors of the glycine transporters GlyT1 and GlyT2 lowered levels of PPIX and markers of oxidative stress selectively in K56211B4 cells, and in primary erythroid cultures from an EPP patient. Our findings open the door to investigation of glycine transport inhibitors for HBS disorders.

Characterization of mesoangioblast cell fate and improved promyogenic potential of a satellite cell-like subpopulation upon transplantation in dystrophic murine muscles.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease caused by the lack of dystrophin in muscle fibers that is currently without curative treatment. Mesoangioblasts (MABs) are multipotent progenitor cells that can differentiate to a myogenic lineage and that can be used to express Dystrophin upon transplantation into muscles, in autologous gene therapy approaches. However, their fate in the muscle environment remains poorly characterized. Here, we investigated the differentiation fate of MABs following their transplantation in DMD murine muscles using a mass cytometry strategy. This allowed the identification and isolation of a fraction of MAB-derived cells presenting common properties with satellite muscle stem cells. This analysis also indicated that most cells did not undergo a myogenic differentiation path once in the muscle environment, limiting their capacity to restore dystrophin expression in transplanted muscles. We therefore assessed whether MAB treatment with cytokines and growth factors prior to engraftment may improve their myogenic fate. We identified a combination of such signals that ameliorates MABs capacity to undergo myogenic differentiation in vivo and to restore dystrophin expression upon engraftment in myopathic murine muscles.

An MFS Transporter-Like ORF from MDR Acinetobacter baumannii AIIMS 7 Is Associated with Adherence and Biofilm Formation on Biotic/Abiotic Surface.

A major facilitator superfamily (MFS) transporter-like open reading frame (ORF) of 453 bp was identified in a pathogenic strain Acinetobacter baumannii AIIMS 7, and its association with adherence and biofilm formation was investigated. Reverse transcription PCR (RT-PCR) showed differential expression in surface-attached biofilm cells than nonadherent cells. In vitro translation showed synthesis of a ~17 kDa protein, further confirmed by cloning and heterologous expression in E. coli DH5α. Up to 2.1-, 3.1-, and 4.1- fold biofilm augmentation was observed on abiotic (polystyrene) and biotic (S. cerevisiae/HeLa) surface, respectively. Scanning electron microscopy (SEM) and gfp-tagged fluorescence microscopy revealed increased adherence to abiotic (glass) and biotic (S. cerevisiae) surface. Extracellular DNA(eDNA) was found significantly during active growth; due to probable involvement of the protein in DNA export, strong sequence homology with MFS transporter proteins, and presence of transmembrane helices. In summary, our findings show that the putative MFS transporter-like ORF (pmt) is associated with adherence, biofilm formation, and probable eDNA release in A. baumannii AIIMS 7.