Lars Aagaard

Identification of endogenous retroviral reading frames in the human genome

BackgroundHuman endogenous retroviruses (HERVs) comprise a large class of repetitive retroelements. Most HERVs are ancient and invaded our genome at least 25 million years ago, except for the evolutionary young HERV-K group. The far majority of the encoded genes are degenerate due to mutational decay and only a few non-HERV-K loci are known to retain intact reading frames. Additional intact HERV genes may exist, since retroviral reading frames have not been systematically annotated on a genome-wide scale.ResultsBy clustering of hits from multiple BLAST searches using known retroviral sequences we have mapped 1.1% of the human genome as retrovirus related. The coding potential of all identified HERV regions were analyzed by annotating viral open reading frames (vORFs) and we report 7836 loci as verified by protein homology criteria. Among 59 intact or almost-intact viral polyproteins scattered around the human genome we have found 29 envelope genes including two novel gammaretroviral types. One encodes a protein similar to a recently discovered zebrafish retrovirus (ZFERV) while another shows partial, C-terminal, homology to Syncytin (HERV-W/FRD).ConclusionsThis compilation of HERV sequences and their coding potential provide a useful tool for pursuing functional analysis such as RNA expression profiling and effects of viral proteins, which may, in turn, reveal a role for HERVs in human health and disease. All data are publicly available through a database at http://www.retrosearch.dk.

B cells and monocytes from patients with active multiple sclerosis exhibit increased surface expression of both HERV-H Env and HERV-W Env, accompanied by increased seroreactivity

BackgroundThe etiology of the neurogenerative disease multiple sclerosis (MS) is unknown. The leading hypotheses suggest that MS is the result of exposure of genetically susceptible individuals to certain environmental factor(s). Herpesviruses and human endogenous retroviruses (HERVs) represent potentially important factors in MS development. Herpesviruses can activate HERVs, and HERVs are activated in MS patients.ResultsUsing flow cytometry, we have analyzed HERV-H Env and HERV-W Env epitope expression on the surface of PBMCs from MS patients with active and stable disease, and from control individuals. We have also analyzed serum antibody levels to the expressed HERV-H and HERV-W Env epitopes. We found a significantly higher expression of HERV-H and HERV-W Env epitopes on B cells and monocytes from patients with active MS compared with patients with stable MS or control individuals. Furthermore, patients with active disease had relatively higher numbers of B cells in the PBMC population, and higher antibody reactivities towards HERV-H Env and HERV-W Env epitopes. The higher antibody reactivities in sera from patients with active MS correlate with the higher levels of HERV-H Env and HERV-W Env expression on B cells and monocytes. We did not find such correlations for stable MS patients or for controls.ConclusionThese findings indicate that both HERV-H Env and HERV-W Env are expressed in higher quantities on the surface of B cells and monocytes in patients with active MS, and that the expression of these proteins may be associated with exacerbation of the disease.

Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity.

IntroductionPreviously, we established a simple method for deriving mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSC-MSCs). These iPSC-MSCs were capable of forming osteogenic structures in scaffolds and nanofibers. The objective of this study is to systematically characterize the mesenchymal characteristics of the iPSC-MSCs by comparing them to bone marrow-derived MSCs (BM-MSCs).MethodsTwo iPSC-MSC lines (named as mRNA-iPSC-MSC-YL001 and lenti-iPSC-MSC-A001) and one BM-MSC line were used for the study. Cell proliferation, presence of mesenchymal surface markers, tri-lineage differentiation capability (osteogenesis, chondrogenesis, adipogenesis), and expression of “stemness” genes were analyzed in these MSC lines.ResultsThe iPSC-MSCs were similar to BM-MSCs in terms of cell morphology (fibroblast-like) and surface antigen profile: CD29+, CD44+, CD73+, CD90+, CD105+, CD11b–, CD14–, CD31–, CD34–, CD45– and HLA-DR–. A faster proliferative capability was seen in both iPSC-MSCs lines compared to the BM-MSCs. The iPSC-MSCs showed adequate capacity of osteogenesis and chondrogenesis compared to the BM-MSCs, while less adipogenic potential was found in the iPSC-MSCs. The iPSC-MSCs and the tri-lineage differentiated cells (osteoblasts, chondrocytes, adipocytes) all lack expression of “stemness” genes: OCT4, SOX2, GDF3, CRIPTO, UTF1, DPPA4, DNMT3B, LIN28a, and SAL4.ConclusionsThe MSCs derived from human iPSCs with our method have advanced proliferation capability and adequate osteogenic and chondrogenic properties compared to BM-MSCs. However, the iPSC-MSCs were less efficient in their adipogenicity, suggesting that further modifications should be applied to our method to derive iPSC-MSCs more closely resembling the naïve BM-MSCs if necessary.

DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1

Myotonic dystrophy type 1 (DM1) is caused by CUG triplet expansions in the 3′ UTR of dystrophia myotonica protein kinase (DMPK) messenger ribonucleic acid (mRNA). The etiology of this multi-systemic disease involves pre-mRNA splicing defects elicited by the ability of the CUG-expanded mRNA to ‘sponge’ splicing factors of the muscleblind family. Although nuclear aggregation of CUG-containing mRNPs in distinct foci is a hallmark of DM1, the mechanisms of their homeostasis have not been completely elucidated. Here we show that a DEAD-box helicase, DDX6, interacts with CUG triplet-repeat mRNA in primary fibroblasts from DM1 patients and with CUG–RNA in vitro. DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci. Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration of MBNL1 in the nucleus. While the level of CUG-expanded mRNA is unaffected by increased DDX6 expression, the mRNA re-localizes to the cytoplasm and its interaction partner MBNL1 becomes dispersed and also partially re-localized to the cytoplasm. Finally, we show that DDX6 unwinds CUG-repeat duplexes in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events.

Adeno-associated virus-delivered polycistronic microRNA-clusters for knockdown of vascular endothelial growth factor in vivo.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in several diseases, including cancer, rheumatoid arthritis and diseases of the eye. Persistent regulation of VEGF by expression of small interfering RNAs targeting VEGF represents a potential future strategy for treatment of such diseases. As a step toward this goal, the present study combines the potency of VEGF‐targeted miRNA mimics, produced from a miRNA cluster, with delivery by adeno‐associated virus (AAV)‐based vectors.

Molecular mechanisms in DM1 — a focus on foci

Myotonic dystrophy type 1 is caused by abnormal expansion of a CTG-trinucleotide repeat in the gene encoding Dystrophia Myotonica Protein Kinase (DMPK), which in turn leads to global deregulation of gene expression in affected individuals. The transcribed mRNA contains a massive CUG-expansion in the 3′ untranslated region (3′UTR) facilitating nucleation of several regulatory RNA-binding proteins, which are thus unable to perform their normal cellular function. These CUG-expanded mRNA–protein aggregates form distinct, primarily nuclear foci. In differentiated muscle cells, most of the CUG-expanded RNA remains in the nuclear compartment, while in dividing cells such as fibroblasts a considerable fraction of the mutant RNA reaches the cytoplasm, consistent with findings that both nuclear and cytoplasmic events are mis-regulated in DM1. Recent evidence suggests that the nuclear aggregates, or ribonuclear foci, are more dynamic than previously anticipated and regulated by several proteins, including RNA helicases. In this review, we focus on the homeostasis of DMPK mRNA foci and discuss how their dynamic regulation may affect disease-causing mechanisms in DM1.

Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors.

Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.

Gene conversion and purifying selection of a placenta-specific ERV-V envelope gene during simian evolution

BackgroundMost human endogenous retroviruses (HERVs) invaded our genome at least 25 million years ago. The majority of the viral genes are degenerated, since no selection preserves them within the genome. However, a few intact and very old HERV genes exist, and likely are beneficial for the host. We here address evolutionary aspects of two HERV-V envelope genes, ENVV1 and ENVV2, located in tandem and containing a long open reading frame.ResultsThe ENVV2 gene is preserved with an intact reading frame during simian evolution, but none of the ENVV genes are found in the prosimian species tested. While we observe many transposon insertions in the gag and pol regions of the ERV-V2 provirus, the ENVV2 genes have escaped transposon crossfire in all species tested. Additional analysis of nucleotide substitutions provides further strong evidence of purifying selection on the ENVV2 gene during primate evolution. The other copy, ENVV1, seems to be involved in gene conversion of the major part of the envelope. Furthermore, ENVV1 and ENVV2 show placenta-specific expression in human and a baboon species.ConclusionOur analyses show that ERV-V entered our genome after the split between simian and prosimian primates. Subsequent purifying selection and gene conversion have preserved two copies of the ENVV envelope gene in most species. This is the first case of gene conversion involving long open reading frames in HERVs. Together with the placenta-specific expression of the human and baboon ENVV1 and ENVV2 envelope genes, these data provide strong evidence of a beneficial role for the host.

Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells

To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1(b) cells with respect to infection though the ecotropic receptor pathway.

A Novel Homologous Model for Gene Therapy of Dwarfism by Non-Viral Transfer of the Mouse Growth Hormone Gene into Immunocompetent Dwarf Mice

The possibilities for non-viral GH gene therapy are studied in immunocompetent dwarf mice (lit/lit). As expression vector we used a plasmid previously employed in immunodeficient dwarf mice (pUBI-hGH-gDNA) by replacing the human GH gene with the genomic sequence of mouse-GH DNA (pUBI-mGH-gDNA). HEK-293 human cells transfected with pUBI-mGH-gDNA produced 3.0 µg mGH/10(6) cells/day compared to 3.7 µg hGH/10(6) cells/day for pUBIhGH- gDNA transfected cells. The weight of lit/lit mice treated with the same two plasmids (50 µg DNA/mouse) by electrotransfer into the quadriceps muscle was followed for 3 months. The weight increase up to 15 days for mGH, hGH and saline treated mice were 0.130, 0.112 and 0.027 g/mouse/day. Most sera from hGH-treated mice contained anti-hGH antibodies already on day 15, with the highest titers on day 45, while no significant anti-mGH antibodies were observed in mGH-treated mice. At the end of 3 months, the weight increase for mGH-treated mice was 34.3%, while the nose-to-tail and femur lengths increased 9.5% and 24.3%. Mouse-GH and hGH circulating levels were 4-5 ng/mL 15 days after treatment, versus control levels of ~0.7 ng GH/mL (P<0.001). In mGH-treated mice, mIGF-I determined on days 15, 45 and 94 were 1.5- to 3-fold higher than the control and 1.2- to 1.6-fold higher than hGH-treated mice. The described homologous model represents an important progress forming the basis for preclinical testing of non-viral gene therapy for GH deficiency.