Loss of a newly discovered microRNA in Chinese hamster ovary cells leads to upregulation of N-glycolylneuraminic acid sialylation on monoclonal antibodies.

Chinese hamster ovary (CHO) cells are known not to express appreciable levels of the sialic acid residue N-glycolylneuraminic acid (NGNA) on monoclonal antibodies. However, we actually have identified a recombinant CHO cell line expressing an IgG with unusually high levels of NGNA sialylation (>30%). Comprehensive multi-OMICs based experimental analyses unraveled the root cause of this atypical sialylation: (1) expression of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene was spontaneously switched on, (2) CMAH mRNA showed an anti-correlated expression to the newly discovered Cricetulus griseus (cgr) specific microRNA cgr-miR-111 and exhibits two putative miR-111 binding sites, (3) miR-111 expression depends on the transcription of its host gene SDK1, and (4) a single point mutation within the promoter region of the sidekick cell adhesion molecule 1 (SDK1) gene generated a binding site for the transcriptional repressor histone H4 transcription factor HINF-P. The resulting transcriptional repression of SDK1 led to a downregulation of its co-expressed miR-111 and hence to a spontaneous upregulation of CMAH expression finally increasing NGNA protein sialylation.

Kinin B1 receptor blockade and ACE inhibition attenuate cardiac postinfarction remodeling and heart failure in rats.

INTRODUCTION: The aim of the present study was to evaluate the effects of the novel kinin B1 receptor antagonist BI113823 on postinfarction cardiac remodeling and heart failure, and to determine whether B1 receptor blockade alters the cardiovascular effects of an angiotensin 1 converting enzyme (ACE) inhibitor in rats. METHODS AND RESULTS: Sprague Dawley rats were subjected to permanent occlusion of the left coronary artery. Cardiovascular function was determined at 6weeks postinfarction. Treatment with either B1 receptor antagonist (BI113823) or an ACE inhibitor (lisinopril) alone or in combination significantly reduced the heart weight-to-body weight and lung weight-to-body weight ratios, and improved postinfarction cardiac function as evidenced by greater cardiac output, the maximum rate of left ventricular pressure rise (±dP/dtmax), left ventricle ejection fraction, fractional shorting, better wall motion, and attenuation of elevated left ventricular end diastolic pressure (LVEDP). Furthermore, all three treatment groups exhibited significant reduction in cardiac interstitial fibrosis, collagen deposition, CD68 positive macrophages, neutrophils, and proinflammatory cytokine production (TNF-α and IL-1ß), compared to vehicle controls. CONCLUSION: The present study shows that treatment with the novel kinin B1 receptor antagonist, BI113823, reduces postinfarction cardiac remodeling and heart failure, and does not influence the cardiovascular effects of the ACE inhibitor.

Efficient propagation of single gene deleted recombinant Sendai virus vectors.

Recombinant Sendai virus vectors (SeVV) have become an attractive tool for basic virological as well as for gene transfer studies. However, to (i) reduce the cellular injury induced by basic recombinant SeV vectors (encoding all six SeV genes as being present in SeV wild-type (wt) genomes) and to (ii) improve SeV vector safety, deletions of viral genes are necessary for the construction of superior SeVV generations. As a strong expression system recombinant replication-incompetent adenoviruses, coding for SeV proteins hemagglutinin-neuraminidase (HN), fusion (F), or matrix (M), were generated and successfully employed for the propagation of single gene deleted (DeltaHN, DeltaF, DeltaM) recombinant SeVV. Further investigations of the propagation procedures required for single gene deleted recombinant SeVV demonstrated (i) modifications of the cell culture medium composition as well as (ii) incubation with vitamin E as crucial steps for the enhancement of SeVV-DeltaHN, -DeltaF, or -DeltaM viral particle yield. Such optimized propagation procedures even led to a successful propagation of HN-deleted viral particles (SeVV-DeltaHN), which has not been reported before.

Cell cycle independent infection and gene transfer by recombinant Sendai viruses.

A common problem for viral vectors in the field of somatic gene therapy is the dependence of an efficient cellular transduction on the cell cycle phase of target cells. An optimized viral vector system should therefore transduce cells in different cell cycle phases equally to improve transduction efficiencies. Recent observations that recombinant Sendai viruses (SeV) can infect a broad range of different tissues suggested SeV to be a good candidate for future gene therapeutic strategies in which dividing and non-dividing cells have to be reached. However, detailed data on the influence of distinct cell cycle phases on the infection of SeV or related viruses are missing. We report that synchronization of NIH 3T3 cells as well as contact inhibition of human fibroblast cells did not exhibit any negative influence on SeV infection rates. Furthermore, different attractive target tissues like human umbilical cord derived cells or primary human hepatocytes can be reached by SeV efficiently. As an important information for further cell cycle studies of paramyxoviruses we discovered surprisingly that the DNA polymerase inhibitor aphidicolin (induces a G(1)/M arrest) functions as an inhibitor of SeV but not of an adenoviral expression vector. In conclusion, the results demonstrate SeV based vector particles to be an ideal tool to reach equally cells coexisting in different cell cycle phases.

Effects of a novel bradykinin B1 receptor antagonist and angiotensin II receptor blockade on experimental myocardial infarction in rats.

BACKGROUND: The aim of the present study was to evaluate the cardiovascular effects of the novel bradykinin B1 receptor antagonist BI-113823 following myocardial infarction (MI) and to determine whether B1 receptor blockade alters the cardiovascular effects of an angiotensin II type 1 (AT1) receptor antagonist after MI in rats. METHODOLOGY/PRINCIPAL FINDINGS: Sprague Dawley rats were subjected to permanent occlusion of the left descending coronary artery. Cardiovascular function was determined at 7 days post MI. Treatment with either B1 receptor antagonist (BI-113823) or AT1 receptor antagonist (irbesartan) alone or in combination improved post-MI cardiac function as evidenced by attenuation of elevated left ventricular end diastolic pressure (LVEDP); greater first derivative of left ventricular pressure (± dp/dt max), left ventricle ejection fraction, fractional shorting, and better wall motion; as we as reductions in post-MI up-regulation of matrix metalloproteinases 2 (MMP-2) and collagen III. In addition, the cardiac up-regulation of B1 receptor and AT1 receptor mRNA were markedly reduced in animals treated with BI 113823, although bradykinin B2 receptor and angiotensin 1 converting enzyme (ACE1) mRNA expression were not significantly affected by B1 receptor blockade. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that treatment with the novel B1 receptor antagonist, BI-113823 improves post-MI cardiac function and does not influence the cardiovascular effects of AT1 receptor antagonist following MI.

Severe impairment of dendritic cell allostimulatory activity by Sendai virus vectors is overcome by matrix protein gene deletion.

Delivery of Ags to dendritic cells (DCs) plays a pivotal role in the induction of efficient immune responses ranging from immunity to tolerance. The observation that certain viral pathogens are able to infect DCs has led to a concept in which applications of recombinant viruses are used for Ag delivery with the potential benefit of inducing potent Ag-specific T cell responses directed against multiple epitopes. As a prerequisite for such an application, the infection of DCs by recombinant viruses should not interfere with their stimulatory capacity. In this context, we could show that an emerging negative-strand RNA viral vector system based on the Sendai virus (SeV) is able to efficiently infect monocyte-derived human DCs (moDCs). However, after infection with SeV wild type, both the response of DCs to bacterial LPS as a powerful mediator of DC maturation and the allostimulatory activity were severely impaired. Interestingly, using various recombinant SeV vectors that were devoid of single viral genes, we were able to identify the SeV matrix (M) protein as a key component in moDC functional impairment after viral infection. Consequently, use of M-deficient SeV vectors preserved the allostimulatory activity in infected moDCs despite an efficient expression of all other virally encoded genes, thereby identifying M-deficient vectors as a highly potent tool for the genetic manipulation of DCs.

Negative-strand RNA viral vectors: intravenous application of Sendai virus vectors for the systemic delivery of therapeutic genes.

Treatment by gene replacement is critical in the field of gene therapy. Suitable vectors for the delivery of therapeutic genes have to be generated and tested in preclinical settings. Recently, extraordinary features for a local gene delivery by Sendai virus vectors (SeVV) have been reported for different tissues. Here we show that direct intravenous application of SeVV in mice is not only feasible and safe, but it results in the secretion of therapeutic proteins to the circulation, for example, human clotting Factor IX (hFIX). In vitro characterization of first-generation SeVV demonstrated that secreted amounts of hFIX were at least comparable to published results for retroviral or adeno-associated viral vectors. Furthermore, as a consideration for application in humans, SeVV transduction led to efficient hFIX synthesis in primary human hepatocytes, and SeVV-encoded hFIX proteins could be shown to be functionally active in the human clotting cascade. In conclusion, our investigations demonstrate for the first time that intravenous administration of negative-strand RNA viral vectors may become a useful tool for the wide area of gene replacement requirements.