Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A.

nefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking

Chitosan-zein nano-in-microparticles Capable of Mediating in vivo Transgene Expression Following Oral Delivery

The oral route is an attractive delivery route for the administration of DNA-based therapeutics, specifically for applications in gene therapy and DNA vaccination. However, oral DNA delivery is complicated by the harsh and variable conditions encountered throughout gastrointestinal (GI) transit, leading to degradation of the delivery vector and DNA cargo, and subsequent inefficient delivery to target cells. In this work, we demonstrate the development and optimization of a hybrid-dual particulate delivery system consisting of two natural biomaterials, zein (ZN) and chitosan (CS), to mediate oral DNA delivery. Chitosan-Zein Nano-in-Microparticles (CS-ZN-NIMs), consisting of core Chitosan/DNA nanoparticles (CS/DNA NPs) prepared by ionic gelation with sodium tripolyphosphate (TPP), further encapsulated in ZN microparticles, were formulated using a water-in-oil emulsion (W/O). The resulting particles exhibited high CS/DNA NP loading and encapsulation within ZN microparticles. DNA release profiles in simulated gastric fluid (SGF) were improved compared to un-encapsulated CS/ DNA NPs. Further, site-specific degradation of the outer ZN matrix and release of transfection competent CS/ DNA NPs occurred in simulated intestinal conditions with CS/DNA NP cores successfully mediating transfection in vitro. Finally, CS-ZN-NIMs encoding GFP delivered by oral gavage in vivo induced the production of anti-GFP IgA antibodies, demonstrating in vivo transfection and expression. Together, these results demonstrate the successful formulation of CS-ZN-NIMs and their potential to improve oral gene delivery through improved protection and controlled release of DNA cargo

Statins stimulate the production of a soluble form of the receptor for advanced glycation end products

The beneficial effects of statin therapy in the reduction of cardiovascular pathogenesis, atherosclerosis, and diabetic complications are well known. The receptor for advanced glycation end products (RAGE) plays an important role in the progression of these diseases. In contrast, soluble forms of RAGE act as decoys for RAGE ligands and may prevent the development of RAGE-mediated disorders. Soluble forms of RAGE are either produced by alternative splicing [endogenous secretory RAGE (esRAGE)] or by proteolytic shedding mediated by metalloproteinases [shed RAGE (sRAGE)]. Therefore we analyzed whether statins influence the production of soluble RAGE. Lovastatin treatment of either mouse alveolar epithelial cells endogenously expressing RAGE or HEK cells overexpressing RAGE caused induction of RAGE shedding, but did not influence secretion of esRAGE from HEK cells overexpressing esRAGE. Lovastatin-induced secretion of sRAGE was also evident after restoration of the isoprenylation pathway, demonstrating a correlation of sterol biosynthesis and activation of RAGE shedding. Lovastatin-stimulated induction of RAGE shedding was completely abolished by a metalloproteinase ADAM10 inhibitor. We also demonstrate that statins stimulate RAGE shedding at low physiologically relevant concentrations. Our results show that statins, due to their cholesterol-lowering effects, increase the soluble RAGE level by inducing RAGE shedding, and by doing this, might prevent the development of RAGE-mediated pathogenesis

CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma

Clinical development of chimeric antigen receptor (CAR)-T-cell therapy has been enabled by advances in synthetic biology, genetic engineering, clinical-grade manufacturing, and complex logistics to distribute the drug product to treatment sites. A key ambition of the CARAMBA project is to provide clinical proof-of-concept for virus-free CAR gene transfer using advanced Sleeping Beauty (SB) transposon technology. SB transposition in CAR-T engineering is attractive due to the high rate of stable CAR gene transfer enabled by optimized hyperactive SB100X transposase and transposon combinations, encoded by mRNA and minicircle DNA, respectively, as preferred vector embodiments. This approach bears the potential to facilitate and expedite vector procurement, CAR-T manufacturing and distribution, and the promise to provide a safe, effective, and economically sustainable treatment. As an exemplary and novel target for SB-based CAR-T cells, the CARAMBA consortium has selected the SLAMF7 antigen in multiple myeloma. SLAMF7 CAR-T cells confer potent and consistent anti-myeloma activity in preclinical assays in vitro and in vivo. The CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial with virus-free CAR-T cells in Europe, and the first clinical trial that uses advanced SB technology worldwide

CC improve secretion of FVIII-BDD, FVIII-BDD-eGFP and FVIII-FL <i>in vitro</i>.

<p>Heterologous CHO cells were incubated with CC at different concentrations. FVIII activity was determined in cell supernatants after 72 h by chromogenic assay. (A) Effect of the following CC on human (h)FVIII-BDD secretion: Betaine (100; 50; 25 mM), ectoine (150; 100; 50 mM), trehalose (150; 100; 50 mM), sorbitol (150; 100; 50 mM), taurine (150; 100; 50 mM), trimethylamine N-oxide (TMAO;50; 25; 12,5 mM) and sodium 4-phenylbutyrate (4-PBA; 2; 0,4 mM). Number of experiments, n = 2. (B) Effect of betaine, ectoine, and the endoplasmatic ATPase inhibitors curcumin and thapsigargin on FVIII-.BDD-eGFP secretion. Butylated hydroxyanisole (BHA) is added as treatment control. n = 3. The mean FVIII secretion level ± SD of untreated hFVIII-BDD-eGFP expressing cells was 19±12 IU per 10e6 cells per 72 h. (C) FVIII-BDD-eGFP secretion into cell supernatants over time at different betaine concentrations. n = 3. (D) Influence of betaine, ectoine, curcumin and thapsigargin on FVIII-FL secretion 72 hours following drug supplementation. n = 3. All values are presented as means ± SEM. ANOVA test * <i>P</i><.05; ** <i>P</i><.001.</p

Rescue of mutant FVIII proteins <i>in vitro</i> and <i>in vivo</i>.

<p>(A–D) HepG2 cells expressing hFVIII muteins were incubated with CC for 72 h. Amount of hFVIII activity in cell supernatant of HepG2 cells expressing hFVIII-BDD Q305P (A and C) or hFVIII-BDD W2313A (B and C) was measured 72 h post betaine treatment. (A and B) show the supplementation of single CC and (C) betaine-ectoine combined treatment. (D) Post CC incubation HepG2 hFVIII-BDDQ305P cells were successively lysed in PBS/0.5% Triton X-100 and PBS/1% SDS. hFVIII antigen was determined in both fractions by indirect ELISA. (E–J) hFVIII-BDDQ305P injected Hem A mice were treated with 2% betaine ad libitum per os in a crossover-study of two groups (each n = 10). After 3 days of treatment hFVIII antigen and activity was measured and treatment was switched between mouse groups. 3 days later, plasma levels were tested again. (E and F) show hFVIII antigen levels, (H and I) the related hFVIII activity levels in plasma of group I or II. (G and J) represent the calculated overall effect of betaine on FVIII antigen levels (G) or FVIII activity (J). square symbols indicate samples of the first measuring point, triangles the second one; clear: tap water-administration (control), filled: 2% betaine administration. (K and L) Endogenous murine FIX levels in all injected FVIII knockout mice (K) and murine FVIII levels of all used FIX knockout mice (L) with and without betaine in the drinking water. Normal mouse levels were set to 100%. Values are presented as means ± SEM. (A–D) ANOVA; (E–H; K–L) Student’s t-test; (I–J) Wilcoxon signed rank test;*<i>P</i><.05, **<i>P</i><.005, ***<i>P</i><.0005.</p

Betaine increases solubility of intracellular FVIII.

<p>CHO cells expressing eGFP-tagged FVIII-BDD protein were incubated with and without betaine. (A,B) Flow cytometry analysis was used to determine the eGFP-signal in untreated cells versus cells treated with betaine or control substance BHA. The mean eGFP intensity (X mean GFP) in the range M1 was used as distinctive parameter. (A) representative histogram after 48 h of treatment and (B) values after 72 h presented as means ± SEM of 3 independent experiments. ANOVA **<i>P</i><.001. (C,D) After 72 h incubation cells were successively lysed in PBS/0.5% Triton X-100 and PBS/1% SDS. (C) FVIII antigen was determined in both fractions by indirect ELISA. (D) Triton X-100-soluble and insoluble fractions were separated on SDS-polyacrylamid gradient gels, and hFVIII light chains (lc), eGFP in hFVIII-single chain (sc) and GAPDH were detected by Western blot. Δ indicates lower band of hFVIII lc doublet.</p