No abstract

Radiotherapy is the cornerstone of palliative treatment for primary bone cancer in animals and metastatic bone cancer in humans. However, the mechanism(s) responsible for pain relief after irradiation is unknown. To identify the mechanism through which radiation treatment decreases bone cancer pain, the effect of radiation on mice with painful bone cancer was studied. Analysis of the effects of a 20-Gy treatment on localized sites of painful bone cancers was performed through assessments of animal behavior, radiographs and histological analysis. The findings indicated that radiation treatment reduced bone pain and supported reduced cancer burden and reduced osteolysis as mechanisms through which radiation reduces bone cancer pain.

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating and ultimately lethal blistering disease caused by mutations to the Col7a1−/− gene. Development of novel cell therapies for the treatment of RDEB would be fostered by having immunodeficient mouse models able to accept human cell grafts; however, immunodeficient models of many genodermatoses such as RDEB are lacking. To overcome this limitation, we combined the clustered regularly interspaced short palindromic repeats and associated nuclease (CRISPR/Cas9) system with microinjection into NOD/SCID IL2rγcnull (NSG) embryos to rapidly develop an immunodeficient Col7a1−/− mouse model of RDEB. Through dose optimization, we achieve F0 biallelic knockout efficiencies exceeding 80%, allowing us to quickly generate large numbers of RDEB NSG mice for experimental use. Using this strategy, we clearly demonstrate important strain-specific differences in RDEB pathology that could underlie discordant results observed between independent studies and establish the utility of this system in proof-of-concept human cellular transplantation experiments. Importantly, we uncover the ability of a recently identified skin resident immunomodulatory dermal mesenchymal stem cell marked by ABCB5 to reduce RDEB pathology and dramatically extend the lifespan of RDEB NSG mice via reduced skin infiltration of inflammatory myeloid derivatives.

The most used treatment for bone cancer pain is radiation; however, the mechanism responsible for analgesia after irradiation is unknown. The mechanistic influence of a single, localized 10-, 20- or 30-Gy dose of radiation on painful behaviors, osteolysis, histopathology and osteoclast number was evaluated in mice with painful femoral sarcomas. Dramatic reductions in pain behaviors (P < 0.05) and osteolysis (P < 0.0001) were seen in mice irradiated with 20 and 30 Gy. Irradiation reduced the tumor area by more than 75% (P < 0.05) but did not affect osteoclast frequency per mm2 tumor. Treatment with 20 Gy prior to tumor injection had no effect on tumor growth or pain behaviors, suggesting that radiation reduces osteolysis and pain through direct tumor effects. To demonstrate that tumor elimination was responsible for reduction in osteolysis and pain, sarcoma cells containing the suicide gene cytosine deaminase (CD) were inoculated into femora. After onset of bone cancer pain, mice were treated with the prodrug 5-fluorocytosine (5-FC). 5-FC treatment significantly reduced both osteolysis (P < 0.0005) and bone cancer pain (P < 0.05). The findings in this study demonstrate that one mechanism through which radiation decreases bone cancer pain is by direct effects on tumor cells.

Recessive dystrophic epidermolysis bullosa (RDEB) is a complex inherited skin disorder caused by loss-of-function mutations in the COL7A1 gene. In order for an effective treatment of this disorder to be realized, both a thorough understanding of the regulation of COL7A1 and an understanding of the underlying nature of the complications of RDEB is needed. Currently, both post-transcriptional regulation of COL7A1 and the underlying causes of fibrosis in RDEB patients are poorly understood. Here, we describe a previously unknown mechanism of regulation by which miR-29 regulates COL7A1 in a complex network: both directly through targeting its 3’ UTR at two distinct seed regions and indirectly through targeting an essential transcription factor required for basal COL7A1 expression, SP1. In turn miR-29 itself is regulated by SP1 activity and TGF-β signaling. RDEB mice express high levels of TGF-β and significantly lower miR-29 when compared to wild-type cohorts. The sustained decrease in miR-29 in RDEB skin leads to an increase of miR-29 target genes expressed in the skin, including pro-fibrotic extracellular matrix collagens. Collectively, we identify miR-29 as an important factor in both regulating COL7A1 and inhibiting TGF-β mediated fibrosis.

Summary Background Revertant mosaicism has been described previously in recessive dystrophic epidermolysis bullosa (RDEB), manifesting as regions of skin with normal mechanical and biological characteristics. Here we report the discovery of revertant dermal fibroblasts, unique in that all other documented cases of revertant mosaicism occur in epidermal keratinocytes. Objectives To determine the cause of revertant mosaicism found in a patient with RDEB from isolated epidermal keratinocytes and dermal fibroblasts in blister and mosaic skin regions. Methods Skin biopsies were taken from blister and mosaic skin regions of a patient with RDEB. Allele identification was confirmed and the type VII collagen (C7) content and COL7A1 expression profile of isolated keratinocytes and fibroblasts was determined. Results Keratinocytes isolated from the mosaic area had a slight increase in C7, although overall expression of COL7A1 was unchanged between blister and mosaic fibroblasts. Differential allele expression was identified in blister and mosaic fibroblasts using targeted RNA sequencing (TREx), where the allele harbouring a point mutation was preferentially expressed over that containing a frameshift mutation. A crossing over event was identified in mosaic fibroblasts that was not present in blister fibroblasts, yielding a functional COL7A1 allele in a subset of cells. Conclusions In documenting a novel case of revertant mosaicism in RDEB, we have identified dermal fibroblasts as having the capacity to correct blistering functionally. We have also pioneered the use of TREx in quantifying allele‐specific expression. Using fibroblasts instead of keratinocytes for RDEB therapies offers advantages in the local and systemic therapy of RDEB. What's already known about this topic? Revertant mosaicism has been previously documented in patients with recessive dystrophic epidermolysis bullosa (RDEB), however, it has only been found in epidermal keratinocytes. What does this study add? We have demonstrated that COL7A1 gene reversion in dermal fibroblasts occurs and is able to form functional skin in a patient with RDEB. Additionally, we have pioneered a new application for targeted RNA sequencing in quantifying allele‐specific expression in fibroblasts and keratinocytes. What is the translational message? This opens up possibilities for using fibroblasts as local and systemic therapy for patients with RDEB.

Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFB ligand/macrophage colonystimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone-and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.