Marlon R. Veldwijk

A combination of granulocyte-colony-stimulating factor (G-CSF) and plerixafor mobilizes more primitive peripheral blood progenitor cells than G-CSF alone: results of a European phase II study

BACKGROUND AIMS Previous studies in xenograft models have shown that human peripheral blood progenitor cells (PBPC) mobilized with the CXCR4 antagonist plerixafor (AMD3100) have a higher bone marrow (BM) reconstitution potential than granulocyte-colony-stimulating factor (G-CSF)-mobilized PBPC. METHODS PBPC obtained during G-CSF-supported mobilization before and after a supplementary administration of AMD3100 from patients with multiple myeloma and non-Hodgkins lymphoma (n=15; phase II study) were investigated for co-expression of primitive and lineage-associated markers, their proliferative activity in vitro and repopulation potential after clinical transplantation. RESULTS A significant increase in primitive CD34+ CD38(-) cells was observed in intraindividual comparisons of all patients after administration of G-CSF+AMD3100 (peripheral blood: median 8-fold, range 2,4-fold - 39-fold) compared with G-CSF alone. Using a long-term culture-initiating cell assay, this increase was confirmed. After transplantation of G-CSF+AMD3100-mobilized PBPC, the time to leukocyte reconstitution > 1 x 10(3)/microL and platelet reconstitution > 2 x 10(4)/microL was 14 (10-19 days) and 13 days (10-15 days), respectively. A complete and stable hematologic reconstitution (platelets > 1.5 x 10(5)/microL) was observed in 91% of all patients within 35 days. CONCLUSIONS An additional application of AMD3100 to a standard G-CSF mobilization regimen leads to a significant increase in primitive PBPC with high repopulation capacity.

Aminoferrocene-Based Prodrugs Activated by Reactive Oxygen Species

Cancer cells generally generate higher amounts of reactive oxygen species than normal cells. On the basis of this difference, prodrugs have been developed (e.g., hydroxyferrocifen), which remain inactive in normal cells, but become activated in cancer cells. In this work we describe novel aminoferrocene-based prodrugs, which, in contrast to hydroxyferrocifen, after activation form not only quinone methides (QMs), but also catalysts (iron or ferrocenium ions). The released products act in a concerted fashion. In particular, QMs alkylate glutathione, thereby inhibiting the antioxidative system of the cell, whereas the iron species induce catalytic generation of hydroxyl radicals. Since the catalysts are formed as products of the activation reaction, it proceeds autocatalytically. The most potent prodrug described here is toxic toward cancer cells (human promyelocytic leukemia (HL-60), IC(50) = 9 μM, and human glioblastoma-astrocytoma (U373), IC(50) = 25 μM), but not toxic (up to 100 μM) toward representative nonmalignant cells (fibroblasts).

Peripheral blood progenitor cell (PBPC) counts during steady-state haemopoiesis enable the estimation of the yield of mobilized PBPC after granulocyte colony-stimulating factor supported cytotoxic chemotherapy: an update on 100 patients

Peripheral blood progenitor cells (PBPC) can be mobilized using chemotherapy and granulocyte colony‐stimulating factor (G‐CSF). We and others previously reported a correlation of steady‐state PBPC counts and the PBPC yield during mobilization in a small group of patients. Here we present data on 100 patients (patients: 25 non‐Hodgkins lymphoma (NHL), five Hodgkins disease, 35 multiple myeloma (MM), 35 solid tumour) which enabled a detailed analysis of determinants of steady‐state PBPC levels and of mobilization efficiency in patient subgroups. Previous irradiation (P = 0.0034) or previous chemotherapy in patients with haematological malignancies (P = 0.0062) led to a depletion of steady‐state PB CD34+ cells. A correlation analysis showed steady‐state PB CD34+ cells (all patients: r = 0.52, P < 0.0001; NHL patients, r = 0.69, P = 0.0003; MM patients: r = 0.66, P = 0.0001) and PB colony‐forming cells can reliably assess the CD34+ cell yield in mobilized PB. In patients with solid tumour a similar trend was observed in mobilization after the first chemotherapy cycle (r = 0.51, P = 0.05) but not if mobilization occurred after the second or further cycle of a sequential dose‐intensified G‐CSF‐supported chemotherapy regimen, when premobilization CD34+ counts were 18‐fold elevated (P = 0.004). When the patients with MM (r = 0.63, P = 0.0008) or with NHL (r = 0.65, P = 0.006) were analysed separately, a highly significant correlation of the steady‐state PB CD34+ cell count to the mean leukapheresis CD34+ cell yield was found, whereas no correlation was observed for patients with a solid tumour. For patients with haematological malignancies estimates could be calculated which, at a specific steady‐state PB CD34+ cell count, could predict with a 95% probability a defined minimum progenitor cell yield. These results enable recognition of patients who mobilize PBPC poorly and may assist selection of patients for novel mobilization regimens.

Differences in functional activity and antigen expression of granulocytes primed in vivo with filgrastim, lenograstim, or pegfilgrastim.

BACKGROUND: Granulocyte–colony‐stimulating factor (G‐CSF) is known to affect functional activity and antigen expression of neutrophil granulocytes. Beside nonglycosylated filgrastim and glycosylated lenograstim, pegylated filgrastim (pegfilgrastim) has recently been introduced for single administration into clinical use.

Generation of efficient human blood progenitor–targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells

OBJECTIVE Currently standard recombinant adeno-associated virus serotype 2(rAAV2)-based vectors lack the efficiency for gene transfer into primary human CD34(+) peripheral blood progenitor cells (PBPC). MATERIALS AND METHODS An advancement in vector development now allows the generation of rAAV capsid mutants that offer higher target cell efficiency and specificity. To increase the gene transfer into hematopoietic progenitor cells, we applied this method for the first time on primary human CD34(+) PBPC cells. RESULTS On a panel of leukemia cell lines (CML/AML), significantly higher gene transfer efficiency of the rAAV capsid mutants (up to 100% gene transfer) was observed compared to standard rAAV2 vectors. A higher transduction efficiency in the imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S and a pronounced difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S were particularly striking. On solid tumor cell lines, on the other hand, rAAV2 was more efficient than the capsid mutants, suggesting an increased specificity of our capsid mutants for hematopoietic progenitor cells. On primary human CD34(+) PBPC significantly higher (up to eightfold; 16% green fluorescent protein-positive) gene transfer could be obtained with the newly generated vectors compared to standard rAAV2 vectors. CONCLUSION These novel vectors may enable efficient gene transfer using rAAV-based vectors into primary human blood progenitor cells for a future clinical application.

Comparison of genetic variation of breast cancer susceptibility genes in Chinese and German populations

Genome-wide association studies (GWAS) identified several genetic risk factors for breast cancer, however, most of them were validated among women of European ancestry. This study examined single-nucleotide polymorphisms (SNPs) contributing to breast cancer in Chinese (984 cases and 2206 controls) and German (311 cases and 960 controls) populations. Eighteen SNPs significantly associated with breast cancer, previously identified in GWAS were genotyped. Twelve SNPs passed quality control and were subjected to statistical analysis. Seven SNPs were confirmed to be significantly associated with breast cancer in the Chinese population, reflecting three independent loci (ESR1, FGFR2, TOX3) and five of these were also confirmed in the German population. The strongest association was identified for rs2046210 in the Chinese (odds ratio (OR)=1.42, 95% confidence interval (CI)=1.28–1.59, P=1.9 × 10−10) and rs3803662 in the German population (OR=1.43, 95% CI=1.17–1.74, P=4.01 × 10−4), located upstream of the ESR1 and TOX3 gene, respectively. For the first time, rs3757318 at 6q25.1, located next to the gene encoding estrogen receptor α (ESR1) was found to be strongly associated with breast cancer (OR=1.33, 95% CI=1.18–1.49, P=1.94 × 10−6) in the Chinese population. The frequency of this variant was markedly lower in the German population and the association was not significant. Despite the genetic differences, essentially the same risk loci were identified in the Chinese and the German populations. Our study suggested the existence of common genetic factors as well as disease susceptibility differences for breast cancer in both populations and highlighted the importance of performing comparison analyses for disease susceptibility within ethnic populations.

Suicide gene therapy of sarcoma cell lines using recombinant adeno-associated virus 2 vectors

Soft-tissue sarcomas are mesenchymal tumors that respond poorly to systemic chemotherapy. Suicide gene therapy may be an alternative treatment strategy. Here we show a high susceptibility of human sarcoma cell lines for recombinant adeno-associated virus 2 (rAAV-2) suicide vectors: connective tissue sarcoma (HS-1), fibrosarcoma (HT-1080), Ewing sarcoma (RD-ES), Askin tumor (SK-N-MC), rhabdomyosarcoma (A-204) and soft-tissue sarcoma (WSKL-1). Several vectors containing the thymidine kinase (TK) gene under the control of either the cytomegalovirus promoter or the elongation-factor 1 alpha (EF1α) promoter were cloned and tested. Higher expression levels of the transgene were observed in the sarcoma lines when using the EF1α-suicide gene-containing vectors. A complete eradication of rAAV-2-EF1α-TK/eGFP (TK/enhanced green fluorescent protein fusion gene)-transduced tumor cells was shown following exposure to ganciclovir (2.5 μg/ml) in vitro, while at this dose level >90% of mock-transduced tumor cells survived. Xenotransplantation tumor models (intraperitoneal, subcutaneous) for the human sarcoma cell line HS-1 were established in nonobese diabetic/severe-combined immunodeficient mice. Mice transplanted with rAAV-2-EF1α-TK/eGFP-transduced and ganciclovir-exposed tumor cells survived >5 months while in the nontransduced group all mice had died approximately 1 month after inoculation. These data hold promise for further development of rAAV-2-based suicide gene therapy of sarcomas.

Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34+ peripheral blood progenitor cells

BACKGROUND AND AIMS Because of their pluripotency, human CD34(+) peripheral blood progenitor cells (PBPC) are targets of interest for the treatment of many acquired and inherited disorders using gene therapeutic approaches. Unfortunately, most current vector systems lack either sufficient transduction efficiency or an appropriate safety profile. Standard single-stranded recombinant adeno-associated virus 2 (AAV2)-based vectors offer an advantageous safety profile, yet lack the required efficiency in human PBPC. METHODS A panel of pseudotyped AAV vectors (designated AAV2/x, containing the vector genome of serotype 2 and capsid of serotype x, AAV2/1-AAV2/6) was screened on primary human granulocyte-colony-stimulating factor (G-CSF)-mobilized CD34(+) PBPC to determine their gene transfer efficacy. Additionally, double-stranded self-complementary AAV (dsAAV) were used to determine possible second-strand synthesis limitations. RESULTS AAV2/6 vectors proved to be the most efficient [12.8% (1.8-25.4%) transgene-expressing PBPC after a single transduction], being significantly more efficient (all P<0.005) than the other vectors [AAV2/2, 2.0% (0.2-7.3%); AAV2/1, 1.3% (0.1-2.9%); others, <; 1% transgene-expressing PBPC]. In addition, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using pseudotyped dsAAV vectors: for dsAAV2/2 [9.3% (8.3-20.3%); P<0.001] and dsAAV2/6 [37.7% (23.6-61.0%); P<0.001) significantly more PBPC expressed the transgene compared with their single-stranded counterparts; for dsAAV2/1, no significant increase could be observed. CONCLUSIONS We have shown that clinically relevant transduction efficiency levels using AAV-based vectors in human CD34(+) PBPC are feasible, thereby offering an efficient alternative vector system for gene transfer into this important target cell population.

Normal-Tissue Radioprotection by Overexpression of the Copper-Zinc and Manganese Superoxide Dismutase Genes

Background and Purpose:Protection of normal tissue against radiation-induced damage may increase the therapeutic ratio of radiotherapy. A promising strategy for testing this approach is gene therapy-mediated overexpression of the copper-zinc (CuZnSOD) or manganese superoxide dismutase (MnSOD) using recombinant adeno-associated viral (rAAV2) vectors. The purpose of this study was to test the modulating effects of the SOD genes on human primary lung fibroblasts (HPLF) after irradiation.Material and Methods:HPLF were transduced with rAAV2 vectors containing cDNA for the CuZnSOD, MnSOD or a control gene. The cells were irradiated (1–6 Gy), and gene transfer efficiency, apoptosis, protein expression/activity, and radiosensitivity measured by the colony-forming assay determined.Results:After transduction, 90.0% ± 6.4% of the cells expressed the transgene. A significant fivefold overexpression of both SOD was confirmed by an SOD activity assay (control: 21.1 ± 12.6, CuZnSOD: 95.1 ± 17.1, MnSOD: 108.5 ± 36.0 U SOD/mg protein) and immunohistochemistry. CuZnSOD and MnSOD overexpression resulted in a significant radioprotection of HPLF compared to controls (surviving fraction [SF] ratio SOD/control > 1): CuZnSOD: 1.18-fold (95% confidence interval [CI]: 1.06–1.32; p = 0.005), MnSOD: 1.23-fold (95% CI: 1.07–1.43; p = 0.01).Conclusion:Overexpression of CuZnSOD and MnSOD in HPLF mediated an increase in clonogenic survival after irradiation compared to controls. In previous works, a lack of radioprotection in SOD-overexpressing tumor cells was observed. Therefore, the present results suggest that rAAV2 vectors are promising tools for the delivery of radioprotective genes in normal tissue.Hintergrund und Ziel:Der Ansatz, Normalgewebszellen gegen bestrahlungsinduzierte Schäden zu schützen, kann möglicherweise die therapeutische Breite strahlentherapeutischer Ansätze erhöhen. Ein potentieller Ansatz wäre die gentherapeutische Überexpression der Kupfer-Zink-(CuZnSOD) oder Mangan-Superoxiddismutase (MnSOD) mit rekombinanten Adeno-assoziierten viralen (rAAV2) Vektoren. Das Ziel dieser Studie war die Bestimmung der bestrahlungsmodulierenden Effekte der SOD-Gene auf humane primäre Lungenfibroblasten (HPLF).Material und Methodik:HPLF wurden mit rAAV2-Vektoren transduziert, die die cDNA für CuZnSOD, MnSOD oder ein Kontrollgen enthielten. Die Zellen wurden mit 1–6 Gy bestrahlt und deren Gentransfereffizienz, Apoptoserate, Proteinexpression/-aktivität und Strahlensensitivität mit einem Koloniebildungsassay bestimmt.Ergebnisse:Nach Transduktion exprimierten 90,0% ± 6,4% der Zellen das entsprechende Transgen. Für beide SOD konnte mittels Immunhistochemie sowie SOD-Aktivitätsassay eine signifikante Überexpression (fünffach; Kontrolle: 21,1 ± 12,6, CuZnSOD: 95,1 ± 17,1, MnSOD: 108,5 ± 36,0 U SOD/mg Gesamtprotein) gezeigt werden. Diese Überexpression führte zu einer signifikanten Radioprotektion der HPLF im Vergleich zu den Kontrollen (Überlebensfraktion-[SF-]Quotient SOD/Kontrolle > 1): CuZnSOD: 1,18-fach (95%-Konfidenzintervall [CI]: 1,06–1,32; p = 0,005), MnSOD: 1,23-fach (95%-CI: 1,07–1,43; p = 0,01).Schlussfolgerung:Im Vergleich zu den Kontrollen führte die Überexpression von CuZnSOD oder MnSOD in HPLF zu einem erhöhten klonogenen Überleben nach Bestrahlung. Da in vorherigen Arbeiten eine mangelnde Radioprotektion nach Überexpression der SOD-Gene in Tumorzellen beobachtet werden konnte, sind rAAV2-Vektoren erfolgversprechend für die Übertragung von radioprotektiven Genen in Normalgewebszellen.

A method for the efficient cellular uptake and retention of small modified gold nanoparticles for the radiosensitization of cells

UNLABELLED Gold nanoparticles (GNP) enhance the absorbance of photons thereby increasing emission of Auger-/photoelectrons in the nm-μm range. Yet, a major disadvantage is their diameter-dependent cellular uptake with an optimum of ~50 nm which may not offer optimal radiosensitization. A method was developed to enhance the uptake of small GNP. GNP (10nm) were linked to DNA and transferred into HeLa cells by transient transfection (GNP-DT). Treatment of cells with GNP-DT resulted in a strong perinuclear focal accumulation, whereas this was dimmer and sparser for GNP-T (lacking DNA) and close to background levels in GNP-treated cells. Only GNP-DT showed a significant radiosensitizing effect (p=0.005) on clonogenic survival using clinically relevant megavolt x-rays. Our novel method markedly increases the uptake/retention and alters the localization of small GNP in cells compared to unmodified GNP. This work finally enables studying the radiosensitizing effects of differentially sized GNP. FROM THE CLINICAL EDITOR In an effort to increase the radiosensitization of HeLa cells, his paper discusses a transient transfection-based method to enhance gold nanoparticle intracellular delivery.