Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (ColVII). ColVII is the main, if not exclusive, component of anchoring fibrils that adhere the epidermis to the dermal layer of the skin. Mutations in COL7A1 lead to defective anchoring fibrils and subsequent perturbed adhesion between the two skin layers resulting in severe skin fragility and constant wounding without ability to heal. A subgroup of RDEB patients harbors nonsense mutations in COL7A1 which create premature termination codons (PTCs) of translation in the mRNA molecule. This leads to mRNA instability and lack of functional protein synthesized. A group of drugs have been reported for their ability to induce "readthrough" and allow for insertion of an amino acid at the site of PTC, thus producing fulllength protein. The mechanism by which these drugs act is currently not known. Here we focus on a PTC read-through drug, amlexanox, previously approved by the FDA for mouth ulcers. Our data show that amlexanox is able to induce full-length synthesis of ColVII in HpV immortalized RDEB patient derived keratinocytes and fibroblasts, as shown by western blotting. After 48 hours of treatment keratinocytes showed up to 14 fold increase in full-length ColVII protein synthesis as compared to non-treated controls. The same treatment in HpV fibroblasts led up to 3 fold increase in ColVII as compared to non-treated controls, or 25% of normal human fibroblasts. While the mechanism by which amlexanox is able to recover protein synthesis from the PTC containing COL7A1 gene is currently unclear, activation of UPF1 by its phosphorylation has been shown to be required for assembly of mRNA degradation machinery. Our data show that amlexanox treatment increases UPF1 phosphorylation, which may be part of the PTC read-through mechanism. In conclusion, we present data to support amlexanox as a read-through agent for the treatment of RDEB in cases with PTC in COL7A1.
Genome editing represents a promising strategy to correct COL7A1 gene mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). Previously, we used programmable nucleases that create double-stranded DNA breaks (DSBs) to repair COL7A1 mutations through homology-directed repair (HDR) with an exogenous repair template. Delivery of this template can be cytotoxic and DSBs induce undesired insertions and deletions (indels) that compete with desired HDR. To overcome these limitations, we used base editors (BE), a CRISPR/Cas9-based system that uses naturally occurring or laboratory-evolved deaminating enzymes to directly convert A>G, C>T, T>C, or G>A. BE does not lead to significant DSBs, obviates the need for a repair template, and typically offers higher editing efficiencies for point mutations than HDR. We used an optimized A>G base editor (ABEmax) for the RDEB causative R185X and R525X nonsense mutations in the COL7A1gene. We delivered ABEmax mRNA with minimal toxicity into primary fibroblasts from two patients with RDEB, and observed mutation correction rates of up to 50% along with concomitant restoration of COL7A1 protein production. Indel occurrence was minimal, with an observed frequency of w2%, consistent with previous studies. Twenty predicted off-target loci were analyzed by high-throughput sequencing. Treatment with the R185X reagent showed no off-target effects, while the R525X candidate showed A>G editing at one exonic off-target site at a frequency of w6% with <0.5% indels. Base-edited human fibroblasts were injected into an immune-deficient mouse model of RDEB, and human COL7A1expressing cells were observed in vivo. 3D bioprinting was used to deposit base-edited fibroblasts in a biopolymer complex that allowed for significant fibroblast expansion in support of a scalable, ex vivo approach for skin graft generation. These findings suggest that an optimized base editing approach may provide an efficient and precise genome editing method for individualized autologous cell therapy for RDEB.
Facial angiofibromas (AF) and fibrous cephalic plaques (FCP) are typical skin lesions in tuberous sclerosis complex (TSC) that contain TSC2-null fibroblast-like cells with overactivation of mTORC1 signaling. To identify paracrine factors important in TSC tumorigenesis, we used transcriptomic analysis of cells incubated with or without the mTOR inhibitor rapamycin (20 nM), using TSC2-null fibroblasts from AFs and FCPs with paired TSC fibroblasts from normalappearing skin from 4 patients. Unbiased hierarchical clustering identified a group of genes whose expression levels were greater in tumor than normal fibroblasts and negatively regulated by rapamycin. CXCL12 was selected for further study since CXCL12 has been implicated in angiogenesis and cancer. CXCL12 protein levels were measured using ELISA in culture supernatants of TSC2-null tumor cells derived from 4 AFs and 3 FCPs as well as the patient normal fibroblasts. CXCL12 protein released into the media by tumor cells over 24 hours averaged 4.6-fold more than patient normal fibroblasts (p¼0.03). Levels of CXCL12 released by FCP cells into media was decreased by 49% with rapamycin (20 nM) treatment for 24 hours. To further study CXCL12 in TSC, we used our mouse model with conditional knockout of Tsc2 in limb bud and ventral skin mesenchyme using homozygous Tsc2 floxed alleles and the Prrx1-cre transgene (termed here Tsc2cKO mice). Tsc2-null dermal fibroblasts isolated from ventral skin of Tsc2cKO mice released 2.9-fold more CXCL12 into media than fibroblasts from control mice. Levels of CXCL12 in the sera of 3.5 weeks old Tsc2cKO mice were 1.7-fold higher than in the sera of control mice (p¼0.01). These results suggest that CXCL12 overexpressed by TSC2-null fibroblasts may serve as a paracrine factor in TSC tumorigenesis.
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