Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment (16K PRL), the underlying molecular mechanisms are poorly understood. We found that 16K PRL induced microRNA-146a (miR146a) expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR146a levels, which resulted in a subsequent decrease in metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a PPCM-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antago-miRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling, as indicated by normal nursing activities. Finally, miR-146a was elevated in the plasma and hearts of PPCM patients, but not in patients with dilated cardiomyopathy. These results demonstrate that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as a biomarker and therapeutic target for PPCM.

Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (< or =24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high levels of transduction (over 80%) and gene expression in vivo after a single exposure to viral supernatant. These results demonstrate that lentiviral vectors are highly effective for gene transfer to human HSC, and that SFFV regulatory sequences can be successfully incorporated to enhance the long-term expression of a transgene in primary human hematopoietic cells in vivo.

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Small interfering RNAs (siRNAs) were designed to target the bcr-abl oncogene, which causes chronic myeloid leukemia (CML) and bcr-abl-positive acute lymphoblastic leukemia (ALL). Chemically synthesized anti-bcr-abl siRNAs were selected using reporter gene constructs and were found to reduce bcr-abl mRNA up to 87% in bcr-abl-positive cell lines and in primary cells from CML patients. This mRNA reduction was specific for bcr-abl because c-abl and c-bcr mRNA levels remained unaffected. Furthermore, protein expression of BCR-ABL and of laminA/C was reduced by specific siRNAs up to 80% in bcr-abl-positive and normal CD34 ؉ cells, respectively. Finally, antibcr-abl siRNA inhibited BCR-ABL-dependent, but not cytokine-dependent, proliferation in a bcr-abl-positive cell line. These data demonstrate that siRNA can specifically and efficiently interfere with the expression of an oncogenic fusion gene in hematopoietic cells. IntroductionRNA interference (RNAi) describes a highly conserved regulatory mechanism that mediates sequence-specific posttranscriptional gene silencing initiated by double-stranded RNA (dsRNA). 1-3 The RNase III enzyme Dicer processes dsRNA into approximately 22-nucleotide (nt) small interfering RNAs (siRNAs) 4 that serve as guide sequences to induce targetspecific mRNA cleavage by the RNA-induced silencing complex RISC. 5 In plants and Caenorhabditis elegans, RNAi may involve the amplification of dsRNA by an RNA-dependent RNA polymerase (RdRP), 6 and it enables systemic, long-term, and heritable gene silencing. In contrast, RNAi in Drosophila and mammals seems cell autonomous, transient, and nonheritable. Because exogenous application of siRNAs can efficiently trigger RNAi in mammalian cells, 7,8 siRNAs are increasingly used in transient (co)transfection assays to modulate gene expression in mammalian cells, including human cells. [9][10][11][12] Fusion transcripts encoding oncogenic proteins may represent potential targets for a tumor-specific RNAi approach. The Philadelphia (Ph) translocation t(9;22)(q34;q11) generates the bcr-abl fusion gene characteristic for chronic myelogenous leukemia (CML) and Phϩ acute lymphoblastic leukemia (ALL). 13 Bcr-abl encodes a constitutively active cytoplasmic tyrosine kinase that is necessary and sufficient to induce and maintain leukemic transformation. [14][15][16] We demonstrate that anti-bcr-abl siRNAs specifically inhibit bcr-abl mRNA expression in hematopoietic cell lines and primary CML cells. They reduce BCR-ABL protein expression and inhibit BCR-ABL-dependent, but not cytokine-dependent, cell proliferation. Therefore, anti-bcr-abl siRNAs may allow further analysis of BCR-ABL functions and, eventually, may lead to RNAi-based therapeutics. Study design siRNAsTwenty-one-nucleotide single-stranded RNAs directed against the fusion sequence of bcr-abl were chemically synthesized (BioSpring, Frankfurt, Germany) ( Figure 1A). The sense and antisense sequences were: b3a2_1, 5Ј-GCAGAGUUCAAAAGCCCUUdTdT-3Ј; b3a2_3, 5Ј-AGCAGAGUUCAAAAGCCCUdTdT-3Ј; and b3a2_1, 5Ј-AAGGGCU...