Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization.

Rapid generation of stable cell lines expressing high levels of erythropoietin, factor VIII, and an antihuman CD20 antibody using lentiviral vectors.

Lentiviral vectors (LVs) are widely recognized as the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Using erythropoietin (EPO), recombinant factor VIII (fVIII), and an anti-CD20 antibody as model proteins, we demonstrate advantages of LV-based gene delivery to achieve high production levels by transduced cells. Highly productive cell clones were able to incorporate up to 100 vector copies per cellular genome, without selection or gene amplification, and were isolated without extensive screening of a large number of clones.

Lentiviral vector-mediated genetic modification of cell substrates for the manufacture of proteins and other biologics.

Transduction with Lentiviral vectors has been shown to be the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Lentiviral vectors have been widely used in research and have recently shown success in clinical trials for human gene therapy. In this paper, we describe the use of lentiviral vectors to generate genetically modified cell substrates for the manufacture of proteins and other complex biologics.

Lentiviral vector platform for production of bioengineered recombinant coagulation factor VIII.

Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100 µg/l of recombinant fVIII, with a per cell production rate of 0.

Animal models of complement-mediated hypersensitivity reactions to liposomes and other lipid-based nanoparticles.

Intravenous injection of some liposomal drugs, diagnostic agents, micelles and other lipid-based nanoparticles can cause acute hypersensitivity reactions (HSRs) in a high percentage (up to 45%) of patients, with hemodynamic, respiratory and cutaneous manifestations. The phenomenon can be explained with activation of the complement (C) system on the surface of lipid particles, leading to anaphylatoxin (C5a and C3a) liberation and subsequent release reactions of mast cells, basophils and possibly other inflammatory cells in blood. These reactions can be reproduced and studied in pigs, dogs and rats, animal models which differ from each other in sensitivity and spectrum of symptoms.

Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments.

HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor.

Poly(amino acid)s: promising enzymatically degradable stealth coatings for liposomes.

Poly(amino acid)s (PAAs) were evaluated as coating polymers for long-circulating liposomes. The pharmacokinetics of PAA-coated liposomes were assessed in rats. Prolonged circulation times were obtained, comparable to those reported for poly(ethylene glycol) (PEG)-liposomes.

Complement activation-related cardiac anaphylaxis in pigs: role of C5a anaphylatoxin and adenosine in liposome-induced abnormalities in ECG and heart function.

Cardiac anaphylaxis is a severe, life-threatening manifestation of acute hypersensitivity reactions to allergens and drugs. Earlier studies highlighted an amplifying effect of locally applied C5a on the process; however, the role of systemic complement (C) activation with C5a liberation in blood has not been explored to date. In the present study, we used the porcine liposome-induced cardiopulmonary distress model for 1) characterizing and quantifying peripheral C activation-related cardiac dysfunction; 2) exploring the role of C5a in cardiac abnormalities and therapeutic potential of C blockage by soluble C receptor type 1 (sCR1) and an anti-C5a antibody (GS1); and 3) elucidating the role of adenosine and adenosine receptors in paradoxical bradycardia, one of the symptoms observed in this model.

Cerebrovascular involvement in liposome-induced cardiopulmonary distress in pigs.

Intravenous administration of liposomes, including Doxil, can cause severe life-threatening hemodynamic changes in pigs. The reaction is due to complement activation, and it is characterized by massive pulmonary hypertension, systemic hypotension, and severe cardiac abnormalities including falling cardiac output, tachy-or bradycardia with arrhythmia. There were no data suggesting the involvement of cerebrovascular changes in this reaction; however, clinical observations allowed this hypothesis.

Inactivation of C5a anaphylatoxin by a peptide that is complementary to a region of C5a.

PL37 (RAARISLGPRCIKAFTE) is an antisense homology box peptide composed of aa 37-53 of C5a-anaphylatoxin and is considered to be the region essential for C5a function. Using a computer program, we designed the complementary peptides ASGAPAPGPAGPLRPMF (Pep-A) and ASTAPARAGLPRLPKFF (Pep-B). Pep-A bound to PL37 and to C5a with very slow dissociation as determined by analysis using surface plasmon resonance, whereas Pep-B failed to bind at all.

Complement activation during hemorrhagic shock and resuscitation in swine.

Activation of the complement (C) cascade is known to play a key role in the adverse immune consequences of hemorrhagic trauma with subsequent shock and resuscitation. However, it is not clear whether hypovolemia per se, without trauma and resuscitation, can also lead to C activation. To address this question, we studied the presence, kinetics, and cause of C activation in a porcine model of hemorrhagic shock and resuscitation in the absence of trauma.

Complement C5a receptor-mediated signaling may be involved in neurodegeneration in Alzheimer's disease.

In our earlier results, we demonstrated that cells expressing the complement C5aR are vulnerable since abnormal activation of C5aR caused apoptosis of these cells. In this study, we demonstrate that activation of C5aR by antisense homology box (AHB) peptides synthesized in multiple antigenic peptide form and representing putative interaction sites of the C5a/C5aR evoked calcium influx in TGW neuroblastoma cells. Dose-dependent inhibition of the response was found when the cells were pretreated with C5a, suggesting that C5aR was involved in this process.

F(ab)'2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins.

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)2-IVIG and irrelevant human monoclonal antibody.

CD59 blocks not only the insertion of C9 into MAC but inhibits ion channel formation by homologous C5b-8 as well as C5b-9.

Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b-9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b-9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59(-) human B-cells, which formed non-selective ion channels with a total conductance of 360 +/- 24 pS as measured at the beginning of the steady-state phase of the inward currents.

Potentiation of liposome-induced complement activation by surface-bound albumin.

Large anionic multilamellar liposomes containing 71% membrane cholesterol (MLV) caused complement (C) activation in human serum in vitro, as reflected in significant rises in S protein-bound terminal complex (SC5b-9) and C3a-desarg levels. Increasing the albumin content in serum by 1-4 g/100 ml led to 50-100% further increase in MLV-induced C activation, while higher amounts of exogenous human serum albumin (HSA) gradually lost the capability to potentiate liposomal C activation. HSA alone had no influence on SC5b-9 formation at any level below 12%.