Ulrich Hacker

In Vivo Synthesis of Tumor Necrosis Factor-α in Healthy Humans after Live Yellow Fever Vaccination

Most knowledge about the synthesis of human tumor necrosis factor (TNF)-alpha and its regulation derives from in vitro studies. The hypothesis that the syndrome of malaise, myalgia, and low fever observed after a vaccination with live attenuated yellow fever virus could be associated with increased synthesis of TNF was investigated. Plasma samples of 15 healthy subjects taken before and until day 2 (until day 11 in 5 of these subjects) after yellow fever vaccination, showed a significant increase of plasma TNF concentration on day 2 after vaccination and a second peak on day 7. Interleukin-1 receptor antagonist (IL-1ra) concentration was significantly elevated in all subjects on day 2 after vaccination. In a control group receiving non-live vaccinations, TNF and IL-1ra concentrations were unchanged. Thus, yellow fever vaccination represents a routine medical intervention that results in a reproducible increase in the plasma concentration of TNF and secondary cytokines. This may serve as a unique model for the study of the regulation of TNF synthesis in vivo.

Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency

Gene therapy is an attractive new approach for the treatment of cancer. Therefore, the development of efficient vector systems is of crucial importance in this field. Different adeno‐associated virus (AAV) serotypes have been characterized so far, which show considerable differences in tissue tropism. Consequently, we aimed to characterize the most efficient serotype for this application.

Engineering the AAV capsid to optimize vector–host-interactions

Adeno-associated viral (AAV) vectors are the most widely used delivery system for in vivo gene therapy. Vectors developed from natural AAV isolates achieved clinical benefit for a number of patients suffering from monogenetic disorders. However, high vector doses were required and the presence of pre-existing neutralizing antibodies precluded a number of patients from participation. Further challenges are related to AAVs tropism that lacks cell type selectivity resulting in off-target transduction. Conversely, specific cell types representing important targets for gene therapy like stem cells or endothelial cells show low permissiveness. To overcome these limitations, elegant rational design- as well as directed evolution-based strategies were developed to optimize various steps of AAVs host interaction. These efforts resulted in next generation vectors with enhanced capabilities, that is increased efficiency of cell transduction, targeted transduction of previously non-permissive cell types, escape from antibody neutralization and off-target free in vivo delivery of vector genomes. These important achievements are expected to improve current and pave the way towards novel AAV-based applications in gene therapy and regenerative medicine.

Proinflammatory genotype of interleukin-1 and interleukin-1 receptor antagonist is associated with ESRD in proteinase 3-ANCA vasculitis patients

BACKGROUND Small-vessel vasculitides are associated with antineutrophil cytoplasmic antibodies (ANCAs). Cytoplasmic ANCAs are targeted mainly against proteinase 3 (PR3), whereas myeloperoxidase (MPO) is the major antigen of perinuclear ANCAs. These relapsing vasculitides show heterogeneous clinical pictures, and disease severity may vary broadly from mild local organ manifestation to acute organ failure (eg, renal failure). We tested whether two cytokine polymorphisms in the interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra) genes, known to determine cytokine secretion, are associated with clinical manifestations and outcome of ANCA-associated vasculitides. METHODS Polymerase chain reaction and restriction fragment length polymorphism analyses were performed to determine polymorphisms in the IL-1beta and IL-1ra genes in 79 patients with PR3-ANCA, 30 patients with MPO-ANCA vasculitis, and 196 healthy controls. RESULTS The frequency of the so-called proinflammatory genotype, characterized by high secretion of IL-1beta and low secretion of its antagonist IL-1ra, was increased significantly in patients with PR3-ANCA with end-stage renal disease. CONCLUSION Patients with a renal manifestation of PR3-ANCA vasculitis have an increased risk for developing end-stage renal disease when carrying the proinflammatory IL-1beta/IL-1ra genotype. Anti-inflammatory therapy specifically antagonizing the proinflammatory effect of IL-1beta may be a promising treatment for patients with Wegeners granulomatosis with renal manifestations.

Tropism-modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy

Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvβ8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.

Autophagy determines efficiency of liver‐directed gene therapy with adeno‐associated viral vectors

Use of adeno‐associated viral (AAV) vectors for liver‐directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)–dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV‐mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short‐term treatment with a single dose of torin 1 significantly increased vector‐mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid‐α‐glucosidase in nonhuman primates. Conclusion: We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver‐directed gene therapy using AAV vectors and showed in a proof‐of‐principle study how this virus–host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252–265).

The hairy cell leukemia cell line Eskol spontaneously synthesizes tumor necrosis factor-α and nitric oxide

Abstract Tumor necrosis factor- α (TNF- α ) and nitric oxide (NO) exert a wide array of immunoregulatory, partly related effects. We examined the production of these two mediators by the human hairy cell leukemia cell line Eskol. Combined cell lysate and supernatant of Eskol cells (0.5×10 6 cells ml −1 ) incubated for 18 h, contained a mean of 1.5 ng ml −1 TNF- α . This spontaneous TNF- α synthesis was enhanced by phorbol ester (PMA) and phytohemagglutinin (PHA) and decreased by dexamethasone. Nitrite, the stable product of NO, accumulated in the supernatant of Eskol cells after prolonged incubation. Maximal nitrite concentrations (range: 0.8–3.5 μM at 2×10 6 cells ml −1 ) were detected after 7 days of incubation. NO production was augmented by PHA and reduced by PMA. The inhibitors of NO synthase N G -monomethyl- l -arginine ( l -NMMA) and aminoguanidine decreased NO synthesis. Simultaneous activation with the proinflammatory cytokines, interferon- γ , interleukin-1 β and TNF- α , increased NO synthesis. These results suggest that NO production in Eskol cells results from inducible NO synthase activity. This is the first direct demonstration of NO formation in human lymphoid cells. The cell line, Eskol, may serve as a model to study regulation of TNF- α and NO synthesis in human B-cell leukemia.

Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity.

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.

Grundlagen der Gentherapie

Nur wenige medizinische Forschungsgebiete erhielten in den letzten Jahren so viel Aufmerksamkeit wie die somatische Gentherapie. Die bahnbrechenden molekularbiologischen Entdeckungen in der zweiten Hälfte des letzten Jahrhunderts, die im Jahr 2001 in der Entschlüsselung weiter Teile des menschlichen Genoms ihren bisherigen Höhepunkt fanden [1, 2], schufen die Voraussetzungen dafür.Heute kann man mit zunehmend ausgefeilten Methoden Gene in Zellen einbringen und diese dadurch neu programmieren. Dennoch wird es einige Jahre dauern, bis eine ausgereifte Gentherapie für viele ansonsten nicht behandelbare Krankheiten zur Verfügung stehen wird. Die im vergangenen Jahr z. B. bei Patienten mit Hämophilie B und schwerer Immunschwäche erzielten klinischen Erfolge [3, 4] werden die Forschung so beflügeln, dass die Gentherapie in den nächsten Jahrzehnten zu einer festen therapeutischen Größe in der Medizin heranwachsen kann.

Grundlagen der Gentherapie Prinzipien und Stand der Entwicklung

Zum ThemaNur wenige medizinische Forschungsgebiete erhielten in den letzten Jahren so viel Aufmerksamkeit wie die somatische Gentherapie. Die bahnbrechenden molekularbiologischen Entdeckungen in der zweiten Hälfte des letzten Jahrhunderts, die im Jahr 2001 in der Entschlüsselung weiter Teile des menschlichen Genoms ihren bisherigen Höhepunkt fanden [1, 2], schufen die Voraussetzungen dafür.Heute kann man mit zunehmend ausgefeilten Methoden Gene in Zellen einbringen und diese dadurch neu programmieren. Dennoch wird es einige Jahre dauern, bis eine ausgereifte Gentherapie für viele ansonsten nicht behandelbare Krankheiten zur Verfügung stehen wird. Die im vergangenen Jahr z. B. bei Patienten mit Hämophilie B und schwerer Immunschwäche erzielten klinischen Erfolge [3, 4] werden die Forschung so beflügeln, dass die Gentherapie in den nächsten Jahrzehnten zu einer festen therapeutischen Größe in der Medizin heranwachsen kann.