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Recently it was shown that several new pseudotyped adenoassociated virus (AAV) vectors support cardioselective expression of transgenes. The molecular mechanisms underlying this propensity for cardiac cell transduction are not well understood. We comparatively analyzed AAV vector attachment, internalization, intracellular trafficking, and nuclear uncoating of recombinant self-complementary (sc) AAV2.2 versus pseudotyped scAAV2.6 vectors expressing green fluorescence protein (GFP) in cells of cardiac origin. In cardiac-derived HL-1 cells and primary neonatal rat cardiomyocytes (PNCMs), expression of GFP increased rapidly after incubation with scAAV2.6-GFP, but remained low after scAAV2.2-GFP. Internalization of scAAV2.6-GFP was more efficient than that of scAAV2.2-GFP. Nuclear translocation was similarly efficient for both, but differential nuclear uncoating rates emerged as a key additional determinant of transduction: 30% of all scAAV2.6-GFP genomes translocated to the nucleus became uncoated within 48 h, but only 16% of scAAV2.2-GFP genomes. In contrast to this situation in cells of cardiac origin, scAAV2.2-GFP displayed more efficient internalization and similar (tumor cell line HeLa) or higher (human microvascular endothelial cell (HMEC)) uncoating rates than scAAV.2.6-GFP in non-cardiac cell types. In summary, both internalization and nuclear uncoating are key determinants of cardiac transduction by scAAV2.6 vectors. Any in vitro screening for the AAV pseudotype most suitable for cardiac gene therapy -which is desirable since it may allow significant reductions in vector load in upcoming clinical trials -needs to quantitate both key steps in transduction.

Pharmacological control is a desirable safety feature of oncolytic adenoviruses (oAdV). It has recently been shown that oAdV replication may be controlled by drug-dependent transcriptional regulation of E1A expression. Here, we present a novel concept that relies on tamoxifen-dependent regulation of E1A activity through functional linkage to the mutated hormone-binding domain of the murine estrogen receptor (Mer). Four different E1A-Mer chimeras (ME, EM, E DNLS M, MEM) were constructed and inserted into the adenoviral genome under control of a lung-specific surfactant protein B promoter. The highest degree of regulation in vitro was seen for the corresponding oAdVs Ad.E DNLS M and Ad.MEM, which exhibited an up to 100-fold higher oAdV replication in the presence as compared with the absence of 4-OH-tamoxifen. Moreover, destruction of nontarget cells was six-and 13-fold reduced for Ad.E DNLS M and Ad.MEM, respectively, as compared with Ad.E. Further investigations supported tamoxifen-dependent regulation of Ad.E DNLS M and Ad.MEM in vivo. Induction of Ad.E DNLS M inhibited growth of H441 lung tumors as efficient as a control oAdV expressing E1A. E DNLS M and the MEM chimeras can be easily inserted into a single vector genome, which extends their application to existing oAdVs and strongly facilitates in vivo application.

Background The evolutionary conserved NEAT1-MALAT1 gene cluster encounters high interest in cardiovascular medicine and oncology. The cluster generates large primary transcripts which remain nuclear, whereas novel tRNA-like transcripts (mascRNA, menRNA) enzymatically generated from these precursors translocate to the cytosol. We previously found that NEAT1 and MALAT1 deficient mice display accelerated atherosclerosis and vascular inflammation due to immune dysfunctions. Methods While the previously investigated mice were deficient in the entire NEAT1 or MALAT1 locus, here we aimed to selectively disrupt only tRNA-like transcripts “menRNA” arising from NEAT1, or “mascRNA” arising from MALAT1. To none of these a biological function has been assigned so far. Both lncRNAs give rise to transcripts of vastly different size (NEAT1: 23kb MENb, 3.7kb MENe, 59nt “menRNA”; MALAT1: 8.3 kb primary, 59nt “mascRNA”), and traditional knockout methods are unable to selectively inactivate one of the small transcripts only. Through CRISPR/Cas9 editing we therefore developed human monocyte-macrophage cell lines with short deletions in the respective tRNA-encoding sequences to disrupt normal menRNA or mascRNA formation, respectively. These editing procedures do not affect transcription of the respective lncRNA parent transcripts, and also not disturb regular formation of the triple-helix structures at their 3'-ends which support stabilization of the respective lncRNAs (Fig. 1). Results We found the tRNA-like transcripts menRNA and mascRNA critically influence innate immunity and angiogenesis. In addition to common anomalies resulting from their selective CRISPR-Cas9 mediated deletion (Fig. 1), there are specific disturbances associated with either Δmasc or Δmen cells (Fig. 2). Both ΔmascRNA and ΔmenRNA human monocytes show profoundly altered ribosomal RNA/protein and tRNA-modifying enzyme expression, display anomalous growth/ angiogenetic factor expression, fundamentally change angiogenetic patterns in co-cultures with human endothelial cells, and have gravely disturbed innate immune responses (LPS, DNA and RNA viruses) (Fig. 1). CRISPR-engineered ΔmenRNA cells share remakable similarities with human post-MI PBMCs, suggesting the NEAT1-menRNA system may significantly contribute to post-MI residual inflammatory risk despite optimal standard therapy (Fig. 2). Conclusions Beyond prior work in knockout mice documenting immune function of the NEAT1-MALAT1 cluster, the current study identifies menRNA and mascRNA as important novel components of human innate immunity with relevance for angiogenetic processes. These data provide a second mechanistic link for the apparent relevance of the NEAT1-MALAT1 gene cluster in cardiovascular and malignant diseases. As prototypes of a novel class of small noncoding RNAs (distinct from miRNAs and siRNAs) they may constitute cytosolic therapeutic targets. FUNDunding Acknowledgement Type of funding sources: Other. Main funding source(s): DZHK Shared Expertise Project/B19-006_SE/FKZ 81X2100257/Transcriptome analysis of circulating immune cells to improve the assessment of prognosis and the response to novel anti-inflammatory treatments after myocardial infarction Figure 1. Common anomalies Figure 2. Specific anomalies