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Background:
Pearson Syndrome (PS) is an ultra-rare disease caused by de-novo mitochondrial DNA (mtDNA) deletions. Patients present at infancy with sideroblastic anemia and later develop a ...multisystem metabolic disorder, leading to death in early or late childhood. No disease-modifying treatments are available for PS. Ex-vivo enrichment of functional mitochondria into various cells has been previously demonstrated, as has inter-cellular mitochondrial transfer. In preclinical models of mitochondrial and lysosomal disorders, hematopoietic stem and progenitor cells (HSCs) have been shown capable of carrying and transferring normal organelles into diseased tissues, thereby altering disease phenotype. Here, we show enrichment of PS-derived HSCs with wild-type mitochondria, a process termed mitochondrial augmentation. We further report on three patients with PS treated with autologous HSCs following ex-vivo mitochondrial augmentation.
Methods:
Diagnosis of PS was confirmed by MLPA and deletion-specific dPCR. Colony formation assays were performed on PS patient-derived HSCs, prior to and after mitochondrial augmentation. HSC mobilization was performed with GCSF alone (n=1) or with plerixafor (n=2) prior to leukapheresis. Autologous CD34+ cells were positively-selected using a CliniMACS system, followed by ex-vivo mitochondrial augmentation of the cells with maternal cryopreserved mitochondria carrying normal mtDNA as confirmed by MLPA. Enriched cells were intravenously infused without conditioning. Level of heteroplasmy (relative normal to deleted mtDNA) was determined by deletion-specific dPCR of DNA from peripheral blood. Patients were followed for a period of up to 1 year including clinical and metabolic evaluations. Adverse events were reported as per CTCAE v4.03. Cellular mitochondrial function was studied on peripheral blood mononuclear cells (PBMCs) by ATP content, O2 consumption and flow cytometry for TMRE (tetramethylrhodamine ethyl ester) and MTG (mitotracker green).
Results:
Three patients were treated with production and safety data available, and in two patients efficacy data is available. PS-patient derived HSCs have a diminished capacity to form colonies in vitro (median, 360 colonies per 5x104 cells vs. 1090 in healthy donors). HSC colony forming capacity increased by an average of 30% after mitochondrial augmentation. Target cell dose (4x106 CD34+ cells/kg) was not reached despite two leukapheresis procedures in patients 1 and 2, who received 1.1 and 1.8 million CD34+ cells/kg recipient, respectively. Patient 3 received 2.8 million cells/kg following a single apheresis. Mitochondrial enrichment in the products was 156%, 162% and 114% for patients 1, 2 and 3. To date, the only treatment-related adverse events noted were leukapheresis related, including anemia, hypocalcemia and alkalosis.
In two patients with more than 3 months follow-up, we observed in vivo mitochondrial enrichment starting 3-4 months after cellular therapy, and throughout the follow-up period (Figure). Metabolic function of PBMCs showed improvement at 5 months post-treatment in lymphocyte ATP content, O2 consumption and TMRE:MTG ratio, indicating improved mitochondrial respiratory capacity. Improvement in mitochondrial heteroplasmy and function was in line with clinical findings. Following cell therapy, no events of metabolic crisis occurred, along with normalization of a pre-treatment negative base excess in patient 1 and ongoing improvement in baseline lactate levels in patient 2. Aerobic ability and fine motor functions were superior compared to baseline in both patients. Importantly, quality of life, as measured by the International Pediatric Mitochondrial Disease Score (IPMDS), was greatly improved after treatment.
Conclusion:
We report a first in human study with a novel form of cellular therapy, mitochondrial augmentation, in which we enrich HSCs with organelles encoding non-mutated version of the mtDNA sequence. We show the ability of mitochondrial augmentation to improve in vitro PS-derived HSC function, and improvement in metabolic determinants, aerobic capacity and quality of life of two patients treated. Together, these preliminary clinical data suggest that mitochondrial augmentation therapy is safe, and may alter the clinical course for patients with mitochondrial deletions/mutations including PS.
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Jacoby:Novartis Israel: Consultancy. Blumkin:Minovia Therapeutics: Employment. Sher:Minovia Therapeutics: Employment. Yivgi Ohana:Minovia Therapeutics: Employment. Toren:Novartis Israel: Consultancy.
Defensive symbiosis is amongst nature's most important interactions shaping the ecology and evolution of all partners involved. The pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), ...harbors one obligatory bacterial symbiont and up to seven different facultative symbionts, some of which are known to protect the aphid from pathogens, natural enemies, and other mortality factors. Pea aphids typically drop off the plant when a mammalian herbivore approaches it to avoid incidental predation. Here, we examined whether bacterial symbionts govern the pea aphid dropping behavior by comparing the bacterial fauna in dropping and nondropping aphids of two A. pisum populations, using two molecular techniques: high-throughput profiling of community structure using 16 S reads sequenced on the Illumina platform, and diagnostic polymerase chain reaction (PCR).We found that in addition to the obligatory symbiont, Buchnera aphidicola, the tested colonies of A. pisum harbored the facultative symbionts Serratia symbiotica, Regiella insecticola and Rickettsia, with no significant differences in infection proportions between dropping and nondropping aphids. While S. symbiotica was detected by both techniques, R. insecticola and Rickettsia could be detected only by diagnostic PCR. We therefore conclude that A. pisum's dropping behavior is not affected by its bacterial symbionts and is possibly affected by other factors.
BACKGROUND PLX-R18 cells are placenta-derived mesenchymal-like stromal cells that have been shown to be efficacious in reducing lethality and alleviating symptoms of hematopoietic subsyndrome of ARS ...in rodent and primate models. They are also studied in phase I clinical trials for treatment of incomplete hematopoietic recovery after hematopoietic cell transplantation. Here we show that PLX-R18 cells are also effective mitigators of the ARS elicited by exposure to high-dose x-ray irradiation. METHODS To assess their therapeutic potential, PLX-R18 cells (2.0x10^6 cells/mouse) were administered to 8-week-old C57BL6/J male mice intramuscularly, 1 and 5 days following total body irradiation (TBI) at 11 Gy, or partial-body irradiation with 40% bone marrow sparing (PBI-BM40) at 14 Gy. Body weight and animal survival were monitored, and gastrointestinal tract histology and blood parameters were analyzed. RESULTS In mice exposed to PBI-BM40, rate of survival in mice treated with PLX-R18 cells (87.5%; n=8) in 8 and 9 days was higher than with vehicle control (50.0%; n=8) (p=0.106). Mean body weight in mice treated with PLX-R18 cells (18.2±0.3 g; n=8) was significantly larger than with vehicle control (16.9±0.3 g; n=8) in 6 days after PBI-BM40 (p=0.0037). Significant reduction of level of white blood cells was observed in mice exposed by PBI-BM40 (2.9±0.3 x 10^3 cells/µl; n=4) at 14 days, compared with no irradiation (10.4±1.1 x 10^3 cells/µl; n=3). Administration of PLX-R18 cells inhibited the reduction of white blood cells at 14 days (5.5±1.8 x 10^3 cells/µl; n=4)(p=0.1668). The administration of PLX-R18 cells was no effect in mice exposed to TBI. CONCLUSIONS The current data suggest that PLX-R18 improves symptoms of GI-ARS. Thus, PLX-R18 cells may be novel agent for treatment of GI-ARS.
Placental progesterone synthesis in humans prevents abortion of the fetus by maintaining uterine quiescence and low myometrial excitability. In rodents, a transient steroidogenic output is observed ...in the trophoblast giant cells during mid-pregnancy. Although the exact role of this locally produced progesterone is not clear, rodent trophoblast giant cells are an important cell model for studying the regulation of placental steroidogenesis. This chapter describes the methods we developed to analyze the regulation of genes involved in progesterone biosynthesis in miniature cultures of primary trophoblast cells from rodents. These genes include cholesterol side chain cleavage cytochrome P450 (P450scc) and its accessory proteins, steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD). To obtain giant cells, uterine implantation sites are sliced in half, and the trophoblast giant cell layers are separated from the surrounding decidua by scraping. Cells can subsequently be separated by gentle enzymatic digestion with trypsin, or collagenase, and plated for further study in vitro. This chapter provides instructions, insights, and comments instrumental for performing in situ visualization of giant cell mRNA and proteins, analyzing enzyme activities, and conducting promoter analyses with a limited number of cells.
The hair follicle is an intricate miniature organ dedicated to the production of the structural hair fiber, which is largely composed of hair keratin (HK) proteins. Many developmental pathways ...contribute to hair follicle development; however, the molecular control of HK genes is still far from being resolved. Because the nuclear factor (NF)-κB pathway is known to be involved in the morphogenesis of the hair follicle, we explored the possibility that it may also regulate HK expression. To this end, we analyzed the effect of p65/RelA, an NF-κB effector, on HK regulatory regions using transient transfections into tissue culture cells. Reporter assays on cells transfected with HK promoter constructs and real-time polymerase chain reaction analysis of endogenous HK gene activity demonstrated that p65 induces transcriptional activation of several HK genes of human and mouse origin, primarily that of
acidic hair keratin 5 (Ha5). Focusing on the highly responsive human
Ha5 gene, we defined the major NF-κB/RelA binding sites in its regulatory region and showed the direct binding of p65 to these sites using gel shift assays. We further show, using immunohistochemistry on human hair follicle sections, that p65 is co-expressed with HKs in the hair shaft compartment and may thus be the effector that mediates the NF-κB pathway's activity, which recently was genetically demonstrated to be active in the same region. Thus, we provide evidence for a previously unknown function of NF-κB in hair formation—direct activation of HK target genes—a function that may shed light on some of the symptoms of ectodermal dysplasias.
The hair follicle is an intricate miniature organ dedicated to the production of the structural hair fiber, which is largely composed of hair keratin (HK) proteins. Many developmental pathways ...contribute to hair follicle development; however, the molecular control of HK genes is still far from being resolved. Because the nuclear factor (NF)-kappaB pathway is known to be involved in the morphogenesis of the hair follicle, we explored the possibility that it may also regulate HK expression. To this end, we analyzed the effect of p65/RelA, an NF-kappaB effector, on HK regulatory regions using transient transfections into tissue culture cells. Reporter assays on cells transfected with HK promoter constructs and real-time polymerase chain reaction analysis of endogenous HK gene activity demonstrated that p65 induces transcriptional activation of several HK genes of human and mouse origin, primarily that of acidic hair keratin 5 (Ha5). Focusing on the highly responsive human Ha5 gene, we defined the major NF-kappaB/RelA binding sites in its regulatory region and showed the direct binding of p65 to these sites using gel shift assays. We further show, using immunohistochemistry on human hair follicle sections, that p65 is co-expressed with HKs in the hair shaft compartment and may thus be the effector that mediates the NF-kappaB pathway's activity, which recently was genetically demonstrated to be active in the same region. Thus, we provide evidence for a previously unknown function of NF-kappaB in hair formation-direct activation of HK target genes-a function that may shed light on some of the symptoms of ectodermal dysplasias.
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