Lentivirus-induced 'Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma.

Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDC-TRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 self-differentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated a simpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.

Airway surface liquid contains endogenous DNase activity which can be activated by exogenous magnesium.

INTRODUCTION: The removal of highly viscous mucus from the airways is an important task in the treatment of chronic lung disease like in cystic fibrosis. The inhalation of recombinant human DNase- I (rhDNase-I) is used to facilitate the removal of tenacious airway secretions in different lung diseases and especially in CF. Little is known about endogenous DNase activity in the airway surface liquid. Therefore, we analysed bronchoalveolar lavage fluid (BAL) and exhaled breath condensate (EBC) for the presence of endogenous DNase activity. METHODS: The degradation of plasmid DNA by BAL from patients who had diagnostic bronchoscopy and bronchoalveolar lavage was analyzed. In a group of CF patients and healthy control volunteers the exhaled breath condensate was obtained and also analyzed for the ability to degrade plasmid DNA. In addition, the ability of magnesium to activate endogenous DNase activity in BAL and exhaled breath condensate was investigated. RESULTS: The analyzed BAL samples degraded plasmid DNA only after preincubation with magnesium. When analyzing the exhaled breath condensate the samples obtained from the healthy volunteers showed no DNase activity even after preincubation with magnesium, whereas in one of the two samples obtained from CF patients we found a DNase activity after preincubation with magnesium. CONCLUSION: Increasing the magnesium concentration in the airway surface liquid by aerosolisation of magnesium solutions or oral magnesium supplements could improve the removal of highly viscous mucus in chronic lung disease by activating endogenous DNase activity.

Retroviral MDR1 gene transfer into marrow-engrafting human peripheral blood progenitor cells results in preferential transgene expression in the immature myeloid compartment rather than in mature myeloid progeny in vivo.

BACKGROUND: The objective of multidrug resistance-1 (MDR1) gene therapy is protection of the myeloid cell lineage. It is therefore important to examine the effect of retroviral transduction on myeloid maturation. Transfer of the human MDR1 gene can confer resistance to a variety of cytostatic drugs. For a safe application in humans it is paramount to follow-up the development of transduced cells. METHODS: We transduced human mobilized peripheral blood progenitor cells (PBPC) with a viral vector containing the human MDR1 cDNA and transplanted the transduced cells into non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. The progeny of the transduced cells was analyzed in detail by flow cytometry. RESULTS: A detailed analysis by four-color flow cytometry showed that MDR1 transgene-expressing CD33+ myeloid cells were preferentially negative for the maturation-associated myeloid markers CD11b and CD10, while the untransduced CD33+ myeloid cells expressed significantly higher proportions of these Ag (P<0.01 each). There was no difference in the expression of B- or T-lymphoid Ag among the MDR1-transduced and untransduced lymphoid cells. DISCUSSION: These data indicate that retroviral MDR1 gene transfer results in preferential P-glycoprotein expression in myeloid progenitor cells, which is the target cell population for myelotoxicity of cytostatic drugs.

Retroviral vector insertions in T-lymphocytes used for suicide gene therapy occur in gene groups with specific molecular functions.

Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.

Interaction of bronchoalveolar lavage fluid with polyplexes and lipoplexes: analysing the role of proteins and glycoproteins.

BACKGROUND: Plasmid DNA complexed with cationic lipids (lipoplexes) or cationic polymers (polyplexes) has been used for gene transfer into the lung. Topical gene administration of lipoplexes or polyplexes into the lung after intratracheal instillation or aerosolisation could cause interaction of the complexes with extracellular substances of the airway surface liquid (ASL). These extracellular interactions might be causal for the observed inefficient transfection rate in vivo after topical administration. Therefore, we studied the impact of bronchoalveolar lavage fluid (BALF) on reporter gene expression mediated by non-viral gene vectors. BALF was considered as a model system to mimic possible interactions of the gene vectors with the ASL. METHODS: BALF was taken from 15 patients who underwent diagnostic bronchoscopy. Lipoplexes and polyplexes were incubated with increasing concentrations of BALF and major components of the BALF such as albumin, mucin and alpha(1)-glycoprotein, as a representative of glycosylated proteins. As cationic polymers, we tested dendrimers (fractured PAMAM) and polyethylenimine 25 kDa (PEI) and, as cationic liposomes, we used Lipofect-AMINE. The effect of BALF on polyplexes and lipoplexes was analysed by transfection experiments, fluorescence-quenching assay, 2-D-gel electrophoresis, SDS-PAGE, DNAse protection assay, size and zeta-potential measurements. RESULTS: BALF inhibited polyplex- and lipoplex-mediated gene transfer. Analysing components of BALF, we found that dendrimer-mediated gene transfer was not inhibited by any specific component. PEI-mediated gene transfer was dose-dependently inhibited by alpha(1)-glycoprotein, slightly inhibited by mucin, but not inhibited in the presence of albumin. Lipoplex-mediated gene transfer was inhibited by mucin at higher concentrations and by albumin, but not by alpha(1)-glycoprotein. 2-D-gel electrophoresis revealed that proteins of the BALF were adsorbed more intensively to lipoplexes than to polyplexes. In addition, mucin and alpha(1)-glycoprotein also adsorbed more intensively to lipoplexes than to polyplexes. Adsorption of BALF components led to a decrease in the positive zeta-potential of lipoplexes and led to a negative zeta-potential of polyplexes. Complement cleavage fragment C3 beta, and in the case of lipoplexes also the C3 alpha fragment, were found among the proteins opsonised on gene vectors. CONCLUSIONS: Our study shows that BALF contains inhibitory components for non-viral gene transfer. We could not detect a specific inhibitory component, but inhibition was most likely due to the change in the surface charge of the gene vectors. Interestingly, there is evidence for complement activation when the route of pulmonary gene vector administration is chosen. Consequently, shielding of gene vectors to circumvent interaction with the ASL environment should be a focus for pulmonary administration in the future.

In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers.

BACKGROUND: Gene transfer into the airways could be of importance for the treatment of chronic lung diseases such as cystic fibrosis. In the past few years several attempts have been made to effectively deliver DNA to the lung using different viral and non-viral vector systems. Viral vectors and cationic lipids have been tested intensively but the properties of cationic polymers such as polyethylenimine (PEI) 25 kDa and fractured polyamidoamine dendrimers to deliver DNA to the airways have not been studied. Surfactant preparations have been shown to influence pulmonary adenoviral and naked plasmid DNA mediated gene transfer in vivo. We investigated the gene delivery efficiency of branched PEI 25 kDa and fractured dendrimers to the murine lung in vivo and also examined the effect of surfactant on PEI 25 kDa mediated gene transfer to the lung. METHODS: Cationic polymer/DNA complexes were prepared in 25 mM HEPES buffer (pH = 7.4) or double distilled water and administered to the lungs of BALB/c mice via cannula intubation. The trachea, left and right lung, heart, liver and esophagus were examined for luciferase activity. Inflammation was assessed by performing standard histology. RESULTS: PEI/DNA complexes showed a high level of luciferase gene expression in the lung. Complexes formed in double distilled water exhibited higher gene expression than complexes formed in 25 mM HEPES buffer (pH 7.4). The optimal N/P ratio was found to be N/P = 10 in double distilled water. Luciferase activity was only detected in the lung and decreased rapidly in a time-dependent manner. The addition of a natural surfactant preparation, Alveofact, slightly reduced gene transfer of branched PEI 25 kDa. Luciferase gene expression obtained by using fractured dendrimers was very low. CONCLUSION: The present study demonstrates that PEI 25 kDa, but not polyamidoamine dendrimers, effectively mediates transient gene transfer to the murine lung after intratracheal intubation. In conclusion, branched PEI 25 kDa was found to be an effective vector for pulmonary gene delivery in vivo, being superior to fractured dendrimers.