Computational fluid dynamics analysis of endoluminal aortic perfusion.

Introduction: In peripheral percutaneous (VA) extracorporeal membrane oxygenation (ECMO) procedures the femoral arteries perfusion route has inherent disadvantages regarding poor upper body perfusion due to watershed. With the advent of new long flexible cannulas an advancement of the tip up to the ascending aorta has become feasible. To investigate the impact of such long endoluminal cannulas on upper body perfusion, a Computational Fluid Dynamics (CFD) study was performed considering different support levels and three cannula positions.

Therapeutic afucosylated monoclonal antibody and bispecific T-cell engagers for T-cell acute lymphoblastic leukemia.

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a poor cure rate for relapsed/resistant patients. Due to the lack of T-cell restricted targetable antigens, effective immune-therapeutics are not presently available and the treatment of chemo-refractory T-ALL is still an unmet clinical need. To develop novel immune-therapy for T-ALL, we generated an afucosylated monoclonal antibody (mAb) (ahuUMG1) and two different bispecific T-cell engagers (BTCEs) against UMG1, a unique CD43-epitope highly and selectively expressed by T-ALL cells from pediatric and adult patients.

Cutting Edge: Differential Fine-Tuning of IL-2- and IL-15-Dependent Functions by Targeting Their Common IL-2/15Rβ/γc Receptor.

Interleukin 2 and IL-15 are two closely related cytokines, displaying important functions in the immune system. They share the heterodimeric CD122/CD132 receptor to deliver their signals within target cells. Their specificity of action is conferred by their α receptor chains, IL-2Rα and IL-15Rα.

A personalized approach to interventional treatment of tricuspid regurgitation: experiences from an acute animal study.

Objectives: Interventional treatment of tricuspid valve disease has so far received little attention due to the anatomical challenges in a thrombogenic surrounding. In the present study, we present an imaging-based, personalized interventional approach to the therapy of tricuspid regurgitation.

Methods: In our porcine model, we used rapid prototyping to build a matrix reproducing the geometry of the right atrium that was previously derived from computer tomography (CT) scans.

A new approach to the interventional therapy of tricuspid regurgitation.

Currently, there are no fully developed interventional approaches for the treatment of tricuspid regurgitation (TR). The aim of this study was to evaluate the feasibility of orthotopic interventional placement of a biological prosthetic valve in the tricuspid position by inserting, with a transvenous approach, a self-expandable valve-bearing stent into the right atrium. Based on findings of computerized tomography (CT), a model of the porcine right heart was obtained.

Isolation and evaluation of novel adeno-associated virus sequences from porcine tissues.

High antigenic compatibility and low toxicity is associated with xenograft transplantation of porcine tissues in immunodeficient human recipients. We hypothesized that adeno-associated viruses (AAVs) of porcine origin could be highly compatible to human tissues and thus of good efficiency and low toxicity for in vivo gene transfer. Porcine tissues were screened by PCR for the presence of AAV using primers designed to bind conserved regions and amplify variable regions of an alignment of several AAV sequences available on GenBank.

Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells.

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines.

Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions.

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells.

Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures.

Various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) were screened for their ability to enhance transient gene expression (TGE) in Human Embryonic Kidney 293-EBNA (HEK293E) cells. The effects in HEK293E cells were compared to those in Chinese Hamster Ovary DG44 (CHO-DG44) cells. The iDNMTs and iHDACs were chosen based on their different cellular activities and mechanisms of action.

High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI.

Recombinant proteins are of great commercial and scientific interest. Yet, most production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility; yet, depending on the transfection protocol, transient transfection faces some bottlenecks such as a priori complex formation, limitations in terms of transfection and production media used and the need for medium exchange prior to and/or after transfection.

High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cells.

Adeno-associated virus (AAV)-based vectors belong to the most promising gene transfer vectors in clinical studies. To provide vector for late-stage clinical trials as well as for a potential commercial phase, a scalable, cGMP-compliant process is required. Nearly all vector production protocols currently approved in Phase I clinical trials rely on AAV production in adherent HEK 293 cells in the presence of serum.

RNA interference-mediated suppression and replacement of human rhodopsin in vivo.

Mutational heterogeneity represents a significant barrier to development of therapies for many dominantly inherited diseases. For example, >100 mutations in the rhodopsin gene (RHO) have been identified in patients with retinitis pigmentosa (RP). The development of therapies for dominant disorders that correct the primary genetic lesion and overcome mutational heterogeneity is challenging.

New perspectives for cancer chemotherapy by genetic protection of haematopoietic cells.

The effectiveness of anti-cancer chemotherapy can be limited by acute suppression of the bone marrow (myelosuppression). There is also a risk of therapy-related secondary haematopoietic malignancy as well as acute and longer term effects in other tissues. Clinical strategies have been established to address some of these problems, particularly toxic effects on the bone marrow (acute myelotoxicity); however, there is still substantial scope for improving the management of chronic toxicity and mutagenicity to haematopoietic cells and collateral damage to non-haematopoietic cells during chemotherapy.

Advances in AAV-mediated gene transfer for the treatment of inherited disorders.

The holy grail of gene therapy is the cure of genetic diseases. To achieve this goal, a vector system is desirable that offers a high level of safety combined with clinical efficacy and versatility in terms of potential applications. Gene therapy vectors based on recombinant adeno-associated viruses (AAVs) meet all of these criteria: They are nonpathogenic, devoid of viral coding sequences, and mediate long-term gene expression in the absence of an immune or inflammatory response.

Impact of splice-site mutations of the human MDR1 cDNA on its stability and expression following retroviral gene transfer.

The multidrug resistance 1 (MDR1) gene transfer to hematopoietic cells for protection against cytotoxic drugs has received considerable attention in gene therapy. However, ectopic expression of MDR1 from retroviral vectors has been hampered by its genetic instability resulting from cryptic splice sites within the cDNA. We have evaluated the efficiency of retroviral MDR1 vectors with introduced mutations of the MDR1 cryptic splice donor (cSD) located at nucleotide +339 and of the cryptic splice acceptor (cSA) at nucleotide +2319 of the cDNA.

In vivo quantitative noninvasive imaging of gene transfer by single-photon emission computerized tomography.

Systems aimed at detecting gene expression noninvasively in vivo are desirable for evaluating the outcome of gene transfer in clinical trials. Several approaches have been exploited using magnetic resonance imaging and spectroscopy ((31)P MRS), positron emission tomography (PET), single-photon emission tomography (SPECT), and detection of bioluminescent signals. An ideal system is based on transfer of a marker gene, the activity of which can be detected against a background from the target tissue without interfering with normal physiology or eliciting an immune response.

Efficient trans-splicing in the retina expands the utility of adeno-associated virus as a vector for gene therapy.

Recombinant vectors based on adeno-associated virus (AAV) can efficiently transduce many different cell types, including cells of the retina, resulting in stable gene expression. A major shortcoming of this vector is its small packaging capacity. A trans-splicing approach, which reconstitutes gene expression from two independent AAV vectors, can be used to overcome the vector's packaging limitations.

Noninvasive gene transfer to the lung for systemic delivery of therapeutic proteins.

This study evaluates the use of vectors based on adeno-associated viruses (AAVs) to noninvasively deliver genes to airway epithelial cells as a means for achieving systemic administration of therapeutic proteins. We intranasally delivered AAV vectors to mice in which the same AAV2 genome encoding a cellular marker was packaged in capsids from AAV1, 2, or 5 (AAV2/1, AAV2/2, or AAV2/5, respectively). Gene expression levels achieved in both airways and alveoli were higher with AAV2/5 than with AAV2/1 and were undetectable with AAV2/2.

Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model.

Recombinant vectors based on adeno-associated virus (AAV) or human immunodeficiency 1 (lentivirus) are promising tools for long term in vivo gene delivery. Their design allows the exchange of capsids or envelopes, respectively, theoretically providing the opportunity to transduce a range of cell types. We constructed AAV vectors encoding enhanced green fluorescent protein (EGFP) within an AAV serotype 2 (AAV2) genome contained in an AAV2, five or one capsid (called AAV2/2, AAV2/5 and AAV2/1, respectively).

A single-step affinity column for purification of serotype-5 based adeno-associated viral vectors.

Here we describe a single-step affinity column for purification of vectors based on adeno-associated virus type 5 (AAV5). A sialic-acid-rich protein called mucin was covalently attached to Sepharose and was found to bind AAV5 vectors. Elution with high salt efficiently recovered highly active vectors of greater purity than what is achieved with CsCl(2) sedimentation.

Novel bicistronic retroviral vector expressing gamma-glutamylcysteine synthetase and the multidrug resistance protein 1 (MRP1) protects cells from MRP1-effluxed drugs and alkylating agents.

We have constructed two retroviral vectors, one expressing multidrug resistance protein 1 (MRP1) alone (SF91MRP) and the other expressing MRP1 and gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione biosynthesis (SF91GCS-MRP). We have utilized the hybrid FMEV (Friend mink cell focus-forming/murine embryonic stem cell virus) backbone, previously shown to be efficient in early hematopoietic cells, even when coexpressing two distinct genes. In SF91GCS-MRP, the cDNAs were combined via an internal ribosomal entry site (IRES) sequence from poliovirus, resulting in a bicistronic mRNA produced via the long terminal repeat (LTR).

Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer.

Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.

Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells.

We describe the functional analysis of a novel retroviral vector, SF91m3, which was designed for improved expression of the in vivo selectable marker, multidrug resistance 1 gene (MDR1), in hematopoietic cells. SF91m3 combines several promising features. The vector backbone lacks viral coding sequences and AUG-start codons 5' of the MDR1 cDNA.

Lack of superinfection interference in retroviral vector producer cells.

Vesicular stomatitis virus G protein (VSV-G)-pseudotyped retroviral vectors have become more feasible for clinical gene transfer protocols since stable tetracycline (tet)-regulated packaging cell lines have become available. Here, we analyzed superinfection interference in VSV-G-pseudotyped and classic amphotropic packaging cell lines. No superinfection interference was observed in VSV-G-pseudotyped packaging cell lines.

Membrane-anchored peptide inhibits human immunodeficiency virus entry.

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.