Targeted Activation of Cystic Fibrosis Transmembrane Conductance Regulator.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The majority of CFTR mutations result in impaired chloride channel function as only a fraction of the mutated CFTR reaches the plasma membrane. The development of a therapeutic approach that facilitates increased cell-surface expression of CFTR could prove clinically relevant. Here, we evaluate and contrast two molecular approaches to activate CFTR expression. We find that an RNA-guided nuclease null Cas9 (dCas9) fused with a tripartite activator, VP64-p65-Rta can activate endogenous CFTR in cultured human nasal epithelial cells from CF patients. We also find that targeting BGas, a long non-coding RNA involved in transcriptionally modulating CFTR expression with a gapmer, induced both strong knockdown of BGas and concordant activation of CFTR. Notably, the gapmer can be delivered to target cells when generated as electrostatic particles with recombinant HIV-Tat cell penetrating peptide (CPP), when packaged into exosomes, or when loaded into lipid nanoparticles (LNPs). Treatment of patient-derived human nasal epithelial cells containing F508del with gapmer-CPP, gapmer-exosomes, or LNPs resulted in increased expression and function of CFTR. Collectively, these observations suggest that CRISPR/dCas-VPR (CRISPR) and BGas-gapmer approaches can target and specifically activate CFTR.

Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death.

Long noncoding RNAs (lncRNAs) interact with other RNAs, DNA and/or proteins to regulate gene expression during development. Erythropoiesis is one developmental process that is tightly controlled throughout life to ensure accurate red blood cell production and oxygen transport to tissues. Thus, homeostasis is critical and maintained by competitive outcomes of pro- and anti-apoptotic pathways. LncRNAs are expressed during blood development; however, specific functions are largely undefined. Here, a culture model of human erythropoiesis revealed that lncRNA Fas-antisense 1 (Fas-AS1 or Saf) was induced during differentiation through the activity of essential erythroid transcription factors GATA-1 and KLF1. Saf was also negatively regulated by NF-κB, where decreasing NF-κB activity levels tracked with increasing transcription of Saf. Furthermore, Saf over-expression in erythroblasts derived from CD34(+) hematopoietic stem/progenitor cells of healthy donors reduced surface levels of Fas and conferred protection against Fas-mediated cell death signals. These studies reveal a novel lncRNA-regulated mechanism that modulates a critical cell death program during human erythropoiesis.

Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is a genetic disorder that results in a multi-organ disease with progressive respiratory decline which leads to premature death. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disrupts the capacity of the protein to function as a channel, transporting chloride ions and bicarbonate across epithelial cell membranes. Small molecule treatments targeted at potentiating or correcting CFTR have shown clinical benefits, but are only effective for a small percentage of individuals with specific CFTR mutations. To overcome this limitation, we engineered stromal-derived mesenchymal stem cells (MSC) and HEK293 cells to produce exosomes containing a novel CFTR Zinc Finger Protein fusion with transcriptional activation domains VP64, P65 and Rta to target the CFTR promoter (CFZF-VPR) and activate transcription. Treatment with CFZF-VPR results in robust activation of CFTR transcription in patient derived Human Bronchial Epithelial cells (HuBEC). We also find that CFZF-VPR can be packaged into MSC and HEK293 cell exosomes and delivered to HuBEC cells to potently activate CFTR expression. Connexin 43 appeared to be required for functional release of CFZF-VPR from exosomes. The observations presented here demonstrate that MSC derived exosomes can be used to deliver a packaged zinc finger activator to target cells and activate CFTR. The novel approach presented here offers a next-generation genetic therapy that may one day prove effective in treating patients afflicted with Cystic fibrosis.

Long noncoding RNA Saf and splicing factor 45 increase soluble Fas and resistance to apoptosis.

In multicellular organisms, cell growth and differentiation is controlled in part by programmed cell death or apoptosis. One major apoptotic pathway is triggered by Fas receptor (Fas)-Fas ligand (FasL) interaction. Neoplastic cells are frequently resistant to Fas-mediated apoptosis, evade Fas signals through down regulation of Fas and produce soluble Fas proteins that bind FasL thereby blocking apoptosis. Soluble Fas (sFas) is an alternative splice product of Fas pre-mRNA, commonly created by exclusion of transmembrane spanning sequences encoded within exon 6 (FasΔEx6). Long non-coding RNAs (lncRNAs) interact with other RNAs, DNA, and proteins to regulate gene expression. One lncRNA, Fas-antisense or Saf, was shown to participate in alternative splicing of Fas pre-mRNA through unknown mechanisms. We show that Saf is localized in the nucleus where it interacts with Fas receptor pre-mRNA and human splicing factor 45 (SPF45) to facilitate alternative splicing and exclusion of exon 6. The product is a soluble Fas protein that protects cells against FasL-induced apoptosis. Collectively, these studies reveal a novel mechanism to modulate this critical cell death program by an lncRNA and its protein partner.

Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis.

This paper describes data related to a research article titled, "Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death" [1]. Long noncoding RNAs (lncRNAs) are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis). Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf) described in the research article. Also included are 5' untranslated sequences (UTR) for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34(+) cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT)18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34(+) cells transduced using mock conditions or with lentivirus particles encoding for Saf.

A system for creating stable cell lines that express a gene of interest from a bidirectional and regulatable herpes simplex virus type 1 promoter.

Expression systems used to study the biological function of a gene of interest can have limited utility due to three major factors: i) weak or heterogeneous gene expression; ii) poorly controlled gene expression; and iii) low efficiencies of stable integration and persistent expression. We envisioned that the ideal system should be tightly controlled and coupled with the ability to efficiently create and identify stable cell lines. Herein, we describe a system based upon a bidirectional Herpes simplex virus type 1 promoter that is naturally responsive to the VP16 transactivator and modified to permit tetracycline-regulated transcription on one side while maintaining constitutive activity on the other side. Incorporation of this element into the Sleeping Beauty transposon resulted in a novel bidirectional system with the capacity for high-efficiency stable integration. Using this system, we created stable cell lines in which expression of a gene of interest was tightly and uniformly controlled across a broad range of levels via a novel combination of doxycycline-sensitive de-repression and VP16-mediated sequence-specific induction. The unique characteristics of this system address major limitations of current methods and provide an excellent strategy to investigate the effects of gene dosing in mammalian models.