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Markus Grompe

Publications and source records attributed to Markus Grompe.

At least 19 recordsLinked to original sources
PubMed2013

Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.

Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials. The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome. Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human-murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes in vivo. As predicted from preclinical and clinical studies, rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly-approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications.

Animals↗
PubMed2007

Slow-onset inhibition of fumarylacetoacetate hydrolase by phosphinate mimics of the tetrahedral intermediate: kinetics, crystal structure and pharmacokinetics.

FAH (fumarylacetoacetate hydrolase) catalyses the final step of tyrosine catabolism to produce fumarate and acetoacetate. HT1 (hereditary tyrosinaemia type 1) results from deficiency of this enzyme. Previously, we prepared a partial mimic of the putative tetrahedral intermediate in the reaction catalysed by FAH co-crystallized with the enzyme to reveal details of the mechanism [Bateman, Bhanumoorthy, Witte, McClard, Grompe and Timm (2001) J. Biol. Chem. 276, 15284-15291]. We have now successfully synthesized complete mimics CEHPOBA {4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate} and COPHPAA {3-[(3-carboxy-2-oxopropyl)hydroxyphosphinyl]acrylate}, which inhibit FAH in slow-onset tight-binding mode with K(i) values of 41 and 12 nM respectively. A high-resolution (1.35 A; 1 A=0.1 nm) crystal structure of the FAH.CEHPOBA complex was solved to reveal the affinity determinants for these compounds and to provide further insight into the mechanism of FAH catalysis. These compounds are active in vivo, and CEHPOBA demonstrated a notable dose-dependent increase in SA (succinylacetone; a metabolite seen in patients with HT1) in mouse serum after repeated injections, and, following a single injection (1 mumol/g; intraperitoneal), only a modest regain of FAH enzyme activity was detected in liver protein isolates after 24 h. These potent inhibitors provide a means to chemically phenocopy the metabolic defects of either HT1 or FAH knockout mice and promise future pharmacological utility for hepatocyte transplantation.

Animals↗
PubMed2006

Myeloid lineage progenitors give rise to vascular endothelium.

Despite an important role in vascular development and repair, the origin of endothelial progenitors remains unknown. Accumulating evidence indicates that cells derived from the hematopoietic system participate in angiogenesis. However, the identity and functional role of these cells remain controversial. Here we show that vascular endothelial cells can differentiate from common myeloid progenitors and granulocyte/macrophage progenitors. Endothelial cells derived from transplanted bone marrow-derived myeloid lineage progenitors expressed CD31, von Willebrand factor, and Tie2 but did not express the hematopoietic markers CD45 and F4/80 or the pericyte markers desmin and smooth muscle actin. Lineage tracing analysis in combination with a Tie2-driven Cre/lox reporter system revealed that, in contrast to bone marrow-derived hepatocytes, bone marrow-derived endothelial cells are not the products of cell fusion. The establishment of both hematopoietic and endothelial cell chimerism after parabiosis demonstrates that circulating cells can give rise to vascular endothelium in the absence of acute radiation injury. Our findings indicate that endothelial cells are an intrinsic component of myeloid lineage differentiation and underscore the close functional relationship between the hematopoietic and vascular systems.

Animals↗
PubMed2006

Bone marrow-derived cells fuse with normal and transformed intestinal stem cells.

Transplanted adult bone marrow-derived cells (BMDCs) have been shown to adopt the phenotype and function of several nonhematopoietic cell lineages and promote tumorigenesis. Beyond its cancer enhancing potential, cell fusion has recently emerged as an explanation of how BMDCs regenerate diseased heptocytes, contribute to Purkinje neurons and skeletal and cardiac muscle cells, and participate in skin and heart regeneration. Although bone marrow-derived epithelial cells also have been observed in the intestine, fusion as a mechanism has not been investigated. Here, we show that transplanted BMDCs fuse with both normal and neoplastic intestinal epithelium. Long-term repopulation by donor-derived cells was detected in all principal intestinal epithelial lineages including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, suggesting that the fusion partners of the BMDCs are long-lived intestinal progenitors or stem cells. Fusion of BMDCs with neoplastic epithelium did not result in tumor initiation. Our findings suggest an unexpected role for BMDCs in both regeneration and tumorigenesis of the intestine.

Animals↗
PubMed2006

Natural gene therapy in monozygotic twins with Fanconi anemia.

Monozygotic twin sisters, with nonhematologic symptoms of Fanconi anemia (FA), were discovered to be somatic mosaics for mutations in the FANCA gene. Skin fibroblasts, but not lymphocytes or committed hematopoietic progenitors, were sensitive to DNA cross-linking agents. Molecular analysis revealed, in skin cells of both twins, a frameshift causing deletion in exon 27 (2555deltaT) and an exon 28 missense mutation (2670G>A/R880Q). The latter resulted in primarily cytoplasmic expression and reduced function of the mutant FANCA (R880Q) protein. Surprisingly, the same acquired exon 30 missense change (2927G>A/E966K) was detected in the hematopoietic cells of both sisters, but not in their fibroblasts, nor in either parent. This compensatory mutation existed in cis with the maternal exon 28 mutation, and it restored function and nuclear localization of the resulting protein. Both sisters have been free of hematologic symptoms for more than 2 decades, suggesting that this de novo mutation occurred prenatally in a single hematopoietic stem cell (HSC) in one twin and that descendants of this functionally corrected HSC, via intra-uterine circulation, repopulated the blood lineages of both sisters. This finding suggests that treating FA patients with gene therapy might require transduction of only a few hematopoietic stem cells.

Active Transport, Cell Nucleus↗
PubMed2006

Sustained phosphorylation of Bid is a marker for resistance to Fas-induced apoptosis during chronic liver diseases.

BACKGROUND & AIMS: Increased rates of apoptosis have been reported to play a role in the pathophysiology of many disorders, including liver diseases. Conversely, genetic mutations that result in impairment of programmed cell death have been associated with cancer development. However, apoptosis resistance can also be the result of nongenetic stress adaptation, as seen in the cancer-prone metabolic liver disease hereditary tyrosinemia. To clarify whether stress-induced apoptosis resistance is a general feature of chronic liver diseases, an animal model of chronic cholestasis was examined. METHODS: Studies were performed with mice before and 2 weeks following bile duct ligation and with Fah-/- and Fah/p21-/- mice before and after NTBC withdrawal. RESULTS: Here we show that bile duct ligation induced profound resistance against Fas monoclonal antibody-mediated hepatocyte death. The apoptosis signaling pathway was blocked downstream of caspase-8 activation and proximal to mitochondrial cytochrome c release. In controls, activation of the Fas receptor resulted in rapid dephosphorylation of Bid and its subsequent cleavage, whereas Bid remained phosphorylated and uncleaved in chronic cholestasis and other models of hepatic apoptosis resistance. CONCLUSIONS: We propose a model in which the phosphorylation status of Bid determines the apoptotic threshold of hepatocytes in vivo. Furthermore, resistance to apoptosis in chronic cholestasis may contribute to the long-term risk of cancer in this setting.

Animals↗
PubMed2006

Gene therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice.

Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice, thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely, a peripheral source of enzyme, established by transplanting ARSA-overexpressing hepatocytes from transgenic donors, failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified, enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall, our data provide a strong rationale for implementing HSPC gene therapy in MLD patients.

Animals↗
PubMed2005

In vivo genetic selection of renal proximal tubules.

Repopulation by transplanted cells can result in effective therapy for several regenerative organs including blood, liver, and skin. In contrast, cell therapies for renal diseases are not currently available. Here we developed an animal model in which cells genetically resistant to a toxic intermediate of tyrosine metabolism, homogentisic acid (HGA), were able to repopulate the damaged proximal tubule epithelium of mice with fumarylacetoacetate hydrolase (Fah) deficiency. HGA resistance was achieved by two independent mechanisms. First, Fah+ transplanted bone marrow cells produced significant replacement of damaged proximal tubular epithelium (up to 50%). The majority of bone marrow-derived epithelial cells were generated by cell fusion, not transdifferentiation. In addition to regeneration by fusion-derived epithelial cells, proximal tubular repopulation was also observed by host epithelial cells, which had lost the homogentisic acid dioxygenase gene. These data demonstrate that extensive regeneration of the renal proximal tubule compartment can be achieved through genetic selection of functional cells.

Acute Disease↗
PubMed2005

Fancd2 functions in a double strand break repair pathway that is distinct from non-homologous end joining.

Fanconi anemia (FA) is a multigenic recessive disease resulting in bone marrow failure and increased cancer susceptibility. Cells from FA patients and mouse models are sensitive to DNA interstrand crosslinks (ICLs) and FA mice are moderately sensitive to ionizing radiation (IR). Both kinds of damage induce DNA double strand breaks (DSBs). To date, nine genes in 11 complementation groups have been identified; however, the precise function of the FA pathway remains unclear. Many of the proteins form a nuclear complex necessary for the mono-ubiquitination of the downstream protein, Fancd2. To further investigate the role of the FA pathway in repair of DSBs, we generated Fancd2(-/-)/Prkdc(sc/sc) double mutant mice. Prkdc(sc/sc) mutant mice have a defect in non-homologous end joining (NHEJ) and are sensitive to IR-induced DNA damage. Double mutant animals and primary cells were more sensitive to IR than either single mutant, suggesting that Fancd2 operates in DSB repair pathway distinct from NHEJ. Fancd2(-/-)/Prkdc(sc/sc) double mutant cells were also more sensitive to DSBs generated by a restriction endonuclease. The role of Fancd2 in DSB repair may account for the moderate sensitivity of FA cells to irradiation and FA cells sensitivity to ICLs that are repaired via a DSB intermediate.

Animals↗
PubMed2005

Heterozygosity for p53 (Trp53+/-) accelerates epithelial tumor formation in fanconi anemia complementation group D2 (Fancd2) knockout mice.

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure and an increased susceptibility to cancer. FA is genetically heterogeneous, consisting of at least 11 complementation groups, FA-A through L, including FA-D1 (BRCA2) and D2. We have previously reported an increased incidence of epithelial tumors in Fancd2 knockout mice. To further investigate the role of the FA pathway in tumor prevention, Fancd2 mutant mice were crossed to mice with a null mutation in the tumor suppressor gene, Trp53. The tumor spectrum in Fancd2(-/-)/Trp53(+/-) mice included sarcomas expected in Trp53 heterozygotes, as well as mammary and lung adenocarcinomas that occur rarely in Trp53 heterozygotes. These tumors occurred earlier than in Fancd2(-/-) control mice. Therefore, the Fancd2(-/-)/Trp53(+/-) mice represent an improved model for the study of adenocarcinoma in FA. In addition, it was found that Fancd2(-/-) mouse embryonic fibroblasts but not Fancd2(-/-)/Trp53(-/-) mouse embryonic fibroblasts arrest following DNA damage. Therefore, Trp53 is required for the S phase checkpoint activation observed in Fancd2 mutant cells. Fancd2(-/-)/Trp53(-/-) cells showed an increase in aneuploidy and had multiple gross chromosomal rearrangements.

Aneuploidy↗
PubMed2005

In vivo correction of murine hereditary tyrosinemia type I by phiC31 integrase-mediated gene delivery.

Phage phiC31 integrase is a site-specific recombinase that mediates efficient integration of circular extrachromosomal DNA into the host genome. Here, the integrase system was used to transfer the fumarylacetoacetate hydrolase (FAH) gene into the liver of mice affected with hereditary tyrosinemia type 1. Approximately 3.6% of transfected hepatocytes experienced an integration event. The absolute frequency of integration was 1/1374. A higher proportion of integrase-transfected FAH+ hepatocytes displayed abnormal morphology (bizarre nuclei, enlarged cells) on day 25 after gene transfer, compared to cells not receiving integrase. The increased frequency of these abnormal cells correlated with the amount of integrase plasmid administered, suggesting some form of integrase toxicity in Fah-/- livers. The abnormal hepatocyte appearance was transient and livers analyzed after longer selection (90 days) showed 60% repopulation with only normal healthy FAH+ hepatocytes. A total of seven different integration sites (accounting for >90% of integration) were identified. Serial transplantation of integrase-corrected hepatocytes to Fah-/- recipients was successful, suggesting long-term viability of corrected cells and persistent gene expression through many rounds of cell division. The stability of transgene expression, relatively high integration frequency, and significant site specificity that characterize the phiC31 integration system suggest that it may have utility in many gene therapy settings.

Animals↗
PubMed2005

Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria.

Phenylalanine homeostasis in mammals is primarily controlled by liver phenylalanine hydroxylase (PAH) activity. Inherited PAH deficiency (phenylketonuria or PKU) leads to hyperphenylalaninemia in both mice and humans. A low level of residual liver PAH activity ensures near-normal dietary protein tolerance with normal serum phenylalanine level, but the precise threshold for normal phenylalanine clearance is unknown. We employed hepatocyte transplantation under selective growth conditions to investigate the minimal number of PAH-expressing hepatocytes necessary to prevent hyperphenylalaninemia in mice. Serum phenylalanine levels remained normal in mice exhibiting nearly complete liver repopulation with PAH-deficient hepatocytes (<5% residual wild-type liver PAH activity). Conversely, transplantation of PAH-positive hepatocytes into PAH-deficient Pah(enu2) mice, a model of human PKU, yielded a significant decrease in serum phenylalanine (<700 muM) when liver repopulation exceeded approximately 5%. These data suggest that restoration of phenylalanine homeostasis requires PAH activity in only a minority of hepatocytes.

Animals↗
PubMed2005

Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.

Recombinant adeno-associated virus (rAAV) vector holds promise for gene therapy. Despite a low frequency of chromosomal integration of vector genomes, recent studies have raised concerns about the risk of rAAV integration because integration occurs preferentially in genes and accompanies chromosomal deletions, which may lead to loss-of-function insertional mutagenesis. Here, by analyzing 347 rAAV integrations in mice, we elucidate novel features of rAAV integration: the presence of hot spots for integration and a strong preference for integrating near gene regulatory sequences. The most prominent hot spot was a harmless chromosomal niche in the rRNA gene repeats, whereas nearly half of the integrations landed near transcription start sites or CpG islands, suggesting the possibility of activating flanking cellular disease genes by vector integration, similar to retroviral gain-of-function insertional mutagenesis. Possible cancer-related genes were hit by rAAV integration at a frequency of 3.5%. In addition, the information about chromosomal changes at 218 integration sites and 602 breakpoints of vector genomes have provided a clue to how vector terminal repeats and host chromosomal DNA are joined in the integration process. Thus, the present study provides new insights into the risk of rAAV-mediated insertional mutagenesis and the mechanisms of rAAV integration.

Animals↗
PubMed2005

Liver repair by intra- and extrahepatic progenitors.

Despite its remarkable capacity for endogenous regeneration, the mammalian liver is vulnerable to a number of chronic or acute conditions that exceed or circumvent the proliferative capabilities of its mature cell complement. Bipotential hepatic progenitors, or "oval cells," have been shown to contribute to organ regeneration under such circumstances, both in human patients and in animal models. These progenitors are attractive agents for cell therapy, but have thus far proven challenging to isolate and manipulate. New reports indicating that transplanted bone marrow cells (BMCs) can also generate hepatocytes and contribute to liver repair have attracted considerable attention, because these cells are familiar and accessible to both clinicians and scientists. Recently, the issue of whether nuclear transfer (via cell fusion between donor BMC and recipient hepatocyte) or previously unrecognized differentiation potential (i.e., plasticity/transdifferentiation of BMC) is the primary origin of donor-derived hepatocytes has generated considerable controversy. In the liver, most evidence supports cell fusion as the key agent in the reversal of hepatopathology. However, regardless of their origin, the frequency of hepatocyte correction events is low. As is the case for the delivery of intrahepatic progenitors, substantial improvements in the understanding of this process will be needed before clinical application becomes practical.

Animals↗
PubMed2005

Bone marrow-derived hepatocytes.

Several independent reports have demonstrated that bone marrow cells can give rise to different hepatic epithelial cells types, including oval cells, hepatocytes and duct epithelium. These observations have resulted in the hypothesis that bone marrow resident stem cells, specifically haematopoietic stem cells, are an important source for liver epithelial cell replacement, particularly during chronic injury. However, the bulk of published data on the role of bone marrow stem cells in liver damage suggest that they do not play a significant physiological role in replacement of epithelial cells in any known form of hepatic injury. Fully functional bone marrow-derived hepatocytes indeed exist, but are extremely rare and are generated by cell fusion, not stem cell differentiation.

Bone Marrow Cells↗