Our group is focused on the correction of inherited immunodeficiencies, in particular Chronic Granulomatous Disease, by gene therapy. As part of this work we develop and test gene transfer vectors, optimize gene transfer protocols into human hematopoietic stem cells and, in collaboration with clinicians in Frankfurt, Zurich and London, implement gene therapy PhaseI/II clinical trials.
In addition we are interested in the mechanism of cell transformation by the fusion oncoprotein AML1/ETO. As part of this work we have defined critical structural components within AML1/ETO which are indispensable for cellular transformation and have developed molecular strategies aiming at the inhibition of AML1/ETO oncogenic activity.
A short summary of our work with the main findings and publications is presented below.
Treatment of Chronic Granulomatous Disease (CGD) by gene therapy
Chronic Granulomatous Disease (CGD) is an inherited immunodeficiency characterized by life threatening bacterial and fungal infections. The underlying causes of the disease are defects in the NADPH oxidase complex which plays an important role in microbial killing by producing reactive oxygen species (ROS). Approximately two thirds of all CGD diseases result from mutations within the X-linked CYBB gene encoding for gp91phox (X-CGD).
In 2004 we transplanted two adolescent X-CGD patients with autologous CD34+ blood stem cells containing functional copies of the gp91phox gene after retroviral-mediated gene transfer. Clear clinical benefits were observed in both patients as demonstrated by the eradication of preexisting therapy-resistant and life-threatening infections (Ott et al., Nat Med 2006). However, the clinical success was weakened by an unexpected expansion of gene transduced cells and the silencing of gp91phox expression. While clonal expansion was dominated by cells containing retroviral insertions into growth promoting genes, predominantly at the MDS1/EVI1 locus, gene silencing was caused by epigenetic down regulation of viral promoter activity. One of the patients died 2.5 years after gene therapy. In addition a retrospective analysis revealed the occurrence of genomic instability and monosomy 7 in a significant percentage of gene marked cells. Most likely, the cause of death was severe sepsis due to an acute infection in the absence of an effective phagocytic defense in combination with disturbed hematopoiesis. Monosomy 7 and genomic instability were also observed in our second patient. This patient was transplanted 45 months after gene therapy with blood stem cells from a matched unrelated donor. Further analyses demonstrated that the development of genomic instability in both patients was a consequence of the transactivation of the EVI1 gene after integration of retroviral vectors in the MDS1/EVI1 gene locus (Stein et al., Nat Med 2010).
Despite the severe adverse effects observed in our trial, the clinical benefits experienced by both patients for more than two years after reinfusion of their autologous gene modified cells are impressive evidence that gene therapy can efficiently provide prolonged health improvements to patients and encourages the further development of gene therapy for the long-term correction of CGD. Therefore, new vectors for the gene therapy of CGD are required in which expression of gp91phox is not only high and stable over time but also restricted to mature myeloid cells. This strategy should protect progenitor and stem cells from enhancer-mediated transactivation effects and also from potential side effects due to the aberrant expression of gp91phox. To this end we have developed novel vectors containing tissue-specific promoters for the expression of gp91phox in myeloid cells. These vectors are promising tools for the correction of CGD by gene therapy.
Several projects related to the findings made during our clinical trial have been initiated. The finding of hot spots for retroviral integration has motivated our interest in defining the molecular basis of preferential retroviral integration at particular genomic regions. Similarly, the definition of the pathways involved in EVI1-mediated genomic instability is crucial for the development of strategies for the treatment of blood disorders in which EVI1 is overexpressed. Finally the development of strategies to avoid transgene silencing will be crucial for the therapeutic success of future clinical trials.
2. Oncogenic components and molecular inhibitors of AML1/ETO transformation
AML1/ETO results from the chromosomal translocation t(8;21) and is one of the most frequent translocation products found in acute myeloid leukemia. The aim of our work is to decipher the leukemogenic components of this transforming fusion protein and to explore novel strategies to interfere with its oncogenic activity on the molecular level.
Chromosomal translocations are frequent events during malignant cell transformation, particularly in leukemogenesis. The translocation t(8;21), one of the most frequent chromosomal anomalies in leukemia, involves the AML1 gene on chromosome 21 and the ETO gene on chromosome 8. Essential for the transforming abilities of AML1/ETO is the oligomerization of AML1/ETO proteins and the formation of high molecular weight complexes (HMWCs). We have investigated the consequences of interfering with the ability of AML1/ETO to form HMWCs. We could show that expression of a polypeptide targeted to the oligomerization domain of AML1/ETO, disrupts HMW complex formation, restores expression of AML1/ETO target genes and reverses the block in myeloid differentiation. Moreover, this polypeptide acts synergistically with known inhibitors of histone deacetylase activity to revert the AML1/ETO induced differentiation block. AML1/ETO transformed cells expressing the polypeptide loose progenitor cell characteristics, enter cell cycle arrest, and undergo cell death (Wichmann et al., Cancer Research 2007). Our data propose the oligomerization domain of ETO as a promising target structure for a molecular intervention in AML1/ETO positive leukemias. Currently novel delivery systems for peptides are being established aiming to interfere with AML1/ETO oligomerization. Among these, bacterial expressed peptides are tested for their inhibitory effects on AML1/ETO-dependent cell proliferation.
In collaboration with Holger Gohlke from the Heinrich-Heine-University Düsseldorf we analyzed the energetic contribution of individual amino acids within the NHR2 domain to AML1/ETO dimer-tetramer transition and found a clustered area of five distinct amino acids with strong contribution to the stability of tetramers. Substitution of these five amino acids abolishes tetramer formation without affecting dimer formation. Similar to AML1/ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of AML1-dependent target genes. AML1/ETO dimers do not block myeloid differentiation, are unable to enhance the self-renewal capacity of hematopoietic progenitors and fail to induce leukemia in a murine transplant model (Wichmann et al., Blood 2010). These data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimer-tetramer interface, suitable for a molecular intervention in t(8;21) leukemias. These results also strongly suggest that disruption of the tetramer into two dimers, instead of four monomers, is sufficient to suppress AML1/ETO oncogenic functions. To test this concept, we derived several α-helical 18-mer peptides addressing different areas on the AML1/ETO dimer interacting surface. These 18-mer peptides were tested for their ability to block tetramer formation in an in vitro tetramerization assay. Interestingly, the 18-mer peptide which addresses the hot spot region shows the strongest inhibitory effect on tetramer formation in a dose depended manner. This 18-mer peptide will serve as a lead structure for the development of small molecule inhibitors.
In a complementary approach we aim to elucidate the protein composition of the high molecular weight complex formed by a leukemogenic truncated isoform of AML1/ETO, AML1/ETOtr. These studies will result in a better understanding of the biology of AML1/ETO function and in further potential target structures in t(8;21) leukemias.