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.

Transcriptional Gene Silencing of the Autism-Associated Long Noncoding RNA MSNP1AS in Human Neural Progenitor Cells.

The long noncoding RNA MSNP1AS (moesin pseudogene 1, antisense) is a functional element that was previously associated with autism spectrum disorder (ASD) with genome-wide significance. Expression of MSNP1AS was increased 12-fold in the cerebral cortex of individuals with ASD and 22-fold in individuals with a genome-wide significantly associated ASD genetic marker on chromosome 5p14.1. Overexpression of MSNP1AS in human neuronal cells caused decreased expression of moesin protein, which is involved in neuronal process stability. In this study, we hypothesize that MSNP1AS knockdown impacts global transcriptome levels. We transfected the human neural progenitor cell line SK- N-SH with constructs that caused a 50% suppression of MSNP1AS expression. After 24 h, cells were harvested for total RNA isolation. Strand-specific RNA sequencing analysis indicated altered expression of 1,352 genes, including altered expression of 318 genes following correction for multiple comparisons. Expression of the OAS2 gene was increased >150-fold, a result that was validated by quantitative PCR. Gene ontology analysis of the 318 genes with altered expression following correction for multiple comparisons indicated that upregulated genes were significantly enriched for genes involved in immune response, and downregulated genes were significantly enriched for genes involved in chromatin remodeling. These data indicate multiple transcriptional and translational functions of MSNP1AS that impact ASD-relevant biological processes. Chromatin remodeling and immune response are biological processes implicated by genes with rare mutations associated with ASD. Our data suggest that the functional elements implicated by association of common genetic variants impact the same biological processes, suggesting a possible shared common molecular pathway of ASD.

Evaluation of the Elements of Short Hairpin RNAs in Developing shRNA-Containing CAR T Cells.

Short hairpin RNAs (shRNAs) have emerged as a powerful tool for gene knockdown in various cellular systems, including chimeric antigen receptor (CAR) T cells. However, the elements of shRNAs that are crucial for their efficacy in developing shRNA-containing CAR T cells remain unclear. In this study, we evaluated the impact of different shRNA elements, including promoter strength, orientation, multiple shRNAs, self-targeting, and sense and antisense sequence composition on the knockdown efficiency of the target gene in CAR T cells. Our findings highlight the importance of considering multiple shRNAs and their orientation to achieve effective knockdown. Moreover, we demonstrate that using a strong promoter and avoiding self-targeting can enhance CAR T cell functionality. These results provide a framework for the rational design of CAR T cells with shRNA-mediated knockdown capabilities, which could improve the therapeutic efficacy of CAR T cell-based immunotherapy.

Engineered extracellular vesicles directed to the spike protein inhibit SARS-CoV-2.

SARS-CoV-2 (CoV-2) viral infection results in COVID-19 disease, which has caused significant morbidity and mortality worldwide. A vaccine is crucial to curtail the spread of SARS-CoV-2, while therapeutics will be required to treat ongoing and reemerging infections of SARS-CoV-2 and COVID-19 disease. There are currently no commercially available effective anti-viral therapies for COVID-19, urging the development of novel modalities. Here, we describe a molecular therapy specifically targeted to neutralize SARS-CoV-2, which consists of extracellular vesicles (EVs) containing a novel fusion tetraspanin protein, CD63, embedded within an anti-CoV-2 nanobody. These anti-CoV-2-enriched EVs bind SARS-CoV-2 spike protein at the receptor-binding domain (RBD) site and can functionally neutralize SARS-CoV-2. This work demonstrates an innovative EV-targeting platform that can be employed to target and inhibit the early stages of SARS-CoV-2 infection.

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.

Exosome-mediated stable epigenetic repression of HIV-1.

Human Immunodeficiency Virus (HIV-1) produces a persistent latent infection. Control of HIV-1 using combination antiretroviral therapy (cART) comes at the cost of life-shortening side effects and development of drug-resistant HIV-1. An ideal and safer therapy should be deliverable in vivo and target the stable epigenetic repression of the virus, inducing a stable "block and lock" of virus expression. Towards this goal, we developed an HIV-1 promoter-targeting Zinc Finger Protein (ZFP-362) fused to active domains of DNA methyltransferase 3 A to induce long-term stable epigenetic repression of HIV-1. Cells were engineered to produce exosomes packaged with RNAs encoding this HIV-1 repressor protein. We find here that the repressor loaded anti-HIV-1 exosomes suppress virus expression and that this suppression is mechanistically driven by DNA methylation of HIV-1 in humanized NSG mouse models. The observations presented here pave the way for an exosome-mediated systemic delivery platform of therapeutic cargo to epigenetically repress HIV-1 infection.

Broadly active zinc finger protein-guided transcriptional activation of HIV-1.

Human immunodeficiency virus type 1 (HIV-1) causes a persistent viral infection resulting in the demise of immune regulatory cells. Clearance of HIV-1 infection results in integration of proviral DNA into the genome of host cells, which provides a means for evasion and long-term persistence. A therapeutic compound that specifically targets and sustainably activates a latent HIV-1 provirus could be transformative and is the goal for the "shock-and-kill" approach to a functional cure for HIV-1. Substantial progress has been made toward the development of recombinant proteins that target specific genomic loci for gene activation, repression, or inactivation by directed mutations. However, most of these modalities are too large or too complex for efficient therapeutic application. We describe here the development and testing of a novel recombinant zinc finger protein transactivator, ZFP-362-VPR, which specifically and potently enhances proviral HIV-1 transcription both in established latency models and activity across different viral clades. Additionally, ZFP-362-VPR-activated HIV-1 reporter gene expression in a well-established primary human CD4+ T cell latency model and off-target pathways were determined by transcriptome analyses. This study provides clear proof of concept for the application of a novel, therapeutically relevant, protein transactivator to purge cellular reservoirs of HIV-1.

The Long Noncoding RNA RPS10P2-AS1 Is Implicated in Autism Spectrum Disorder Risk and Modulates Gene Expression in Human Neuronal Progenitor Cells.

Most of the genetic risk for autism spectrum disorder (ASD) is inherited as common genetic variants, although some rare mutations have been identified in individuals with ASD. Common genetic variants are most parsimoniously identified by genome wide association studies. Genome wide association studies have identified several genetic loci with genome wide association with ASD. However, genome wide association studies only identify regions of the genome associated with phenotypic traits. Identification of the functional elements requires additional experimental evidence. Here, we demonstrate that a genome wide association study locus for ASD on chromosome 20p12.1, rs4141463, implicates a noncoding RNA as a functional element. Although rs4141463 lies within an intron of the protein-coding MACROD2 (MACRO domain containing 2) gene, expression of MACROD2 is neither altered in postmortem temporal cortex of individuals with ASD nor correlated with rs4141463 genotype. Our bioinformatics approaches revealed a noncoding RNA transcript near the autism susceptibility signal, RPS10P2-AS1 (ribosomal protein S10 pseudogene 2 anti-sense 1). In a panel of 15 human tissues, RPS10P2-AS1 was expressed at higher levels than the protein-coding MACROD2 in both fetal temporal cortex and adult peripheral blood. In postmortem temporal cortex, expression of RPS10P2-AS1 was increased 7-fold in individuals with ASD (P = 0.02) and increased 8-fold in individuals with the ASD-associated rs4141463 genotype (P = 0.01). Further, RPS10P2-AS1 expression was increased in human neural progenitor cells exposed to model air pollutants, indicating that both genetic and environmental factors that contribute to ASD increased RPS10P2-AS1 expression. Overexpression of RPS10P2-AS1 in human neural progenitor cells indicated substantial changes in neuronal gene expression. These data indicate that genome-wide significant associations with ASD implicate long noncoding RNAs. Because long noncoding RNAs are more abundant in human brain than protein-coding RNAs, this class of molecules is likely to contribute to ASD risk.

Impact of the Autism-Associated Long Noncoding RNA MSNP1AS on Neuronal Architecture and Gene Expression in Human Neural Progenitor Cells.

We previously identified the long noncoding RNA (lncRNA) MSNP1AS (moesin pseudogene 1, antisense) as a functional element revealed by genome wide significant association with autism spectrum disorder (ASD). MSNP1AS expression was increased in the postmortem cerebral cortex of individuals with ASD and particularly in individuals with the ASD-associated genetic markers on chromosome 5p14.1. Here, we mimicked the overexpression of MSNP1AS observed in postmortem ASD cerebral cortex in human neural progenitor cell lines to determine the impact on neurite complexity and gene expression. ReNcell CX and SK-N-SH were transfected with an overexpression vector containing full-length MSNP1AS. Neuronal complexity was determined by the number and length of neuronal processes. Gene expression was determined by strand-specific RNA sequencing. MSNP1AS overexpression decreased neurite number and neurite length in both human neural progenitor cell lines. RNA sequencing revealed changes in gene expression in proteins involved in two biological processes: protein synthesis and chromatin remodeling. These data indicate that overexpression of the ASD-associated lncRNA MSNP1AS alters the number and length of neuronal processes. The mechanisms by which MSNP1AS overexpression impacts neuronal differentiation may involve protein synthesis and chromatin structure. These same biological processes are also implicated by rare mutations associated with ASD, suggesting convergent mechanisms.

Noncoding RNA in the transcriptional landscape of human neural progenitor cell differentiation.

Increasing evidence suggests that noncoding RNAs play key roles in cellular processes, particularly in the brain. The present study used RNA sequencing to identify the transcriptional landscape of two human neural progenitor cell lines, SK-N-SH and ReNcell CX, as they differentiate into human cortical projection neurons. Protein coding genes were found to account for 54.8 and 57.0% of expressed genes, respectively, and alignment of RNA sequencing reads revealed that only 25.5-28.1% mapped to exonic regions of the genome. Differential expression analysis in the two cell lines identified altered gene expression in both protein coding and noncoding RNAs as they undergo neural differentiation with 222 differentially expressed genes observed in SK-N-SH cells and 19 differentially expressed genes in ReNcell CX. Interestingly, genes showing differential expression in SK-N-SH cells are enriched in genes implicated in autism spectrum disorder, but not in gene sets related to cancer or Alzheimer's disease. Weighted gene co-expression network analysis (WGCNA) was used to detect modules of co-expressed protein coding and noncoding RNAs in SK-N-SH cells and found four modules to be associated with neural differentiation. These modules contain varying levels of noncoding RNAs ranging from 10.7 to 49.7% with gene ontology suggesting roles in numerous cellular processes important for differentiation. These results indicate that noncoding RNAs are highly expressed in human neural progenitor cells and likely hold key regulatory roles in gene networks underlying neural differentiation and neurodevelopmental disorders.

A noncoding RNA antisense to moesin at 5p14.1 in autism.

People with autism spectrum disorder (ASD) are characterized by deficits in social interaction, language, and behavioral flexibility. Rare mutations and copy number variations have been identified in individuals with ASD, but in most patients, the causal variants remain unknown. A genome-wide association study (GWAS), designed to identify genes and pathways that contribute to ASD, indicated a genome-wide significant association of ASD with the single-nucleotide polymorphism (SNP) rs4307059 (P = 10⁻¹°), which is located in a gene-poor region of chromosome 5p14.1. We describe here a 3.9-kb noncoding RNA that is transcribed from the region of the chromosome 5p14.1 ASD GWAS association SNP. The noncoding RNA was encoded by the opposite (antisense) strand of moesin pseudogene 1 (MSNP1), and we therefore designated it as MSNP1AS (moesin pseudogene 1, antisense). Chromosome 5p14.1 MSNP1AS was 94% identical and antisense to the X chromosome transcript of MSN, which encodes a protein (moesin) that regulates neuronal architecture. Individuals who carry the ASD-associated rs4307059 T allele showed increased expression of MSNP1AS. The MSNP1AS noncoding RNA bound to MSN, was highly overexpressed (12.7-fold) in postmortem cerebral cortex of individuals with ASD, and could regulate levels of moesin protein in human cell lines. These data reveal a biologically functional element that may contribute to ASD risk.