Impairing MLL-fusion gene-mediated transformation by dissecting critical interactions with the lens epithelium-derived growth factor (LEDGF/p75).

The lens epithelium-derived growth factor (LEDGF/p75) tethers the mixed-lineage leukemia (MLL1) protein complex to chromatin. Likewise, LEDGF/p75 tethers the HIV-1 pre-integration complex to chromatin. We previously demonstrated that expression of the C-terminal fragment fused to enhanced green fluorescent protein (eGFP) (eGFP-LEDGF(325-530)) impaired HIV-1 replication. Here, we explored this strategy to selectively interfere with the leukemogenic activity of MLL-fusion proteins. We found that expression of LEDGF(325-530) impaired the clonogenic growth of MLL-fusion gene transformed human and mouse hematopoietic cells, without affecting the growth of control cells immortalized by the FLT3-ITD mutant or normal lineage-marker-depleted murine bone marrow cells. Expression of LEDGF(325-530) was associated with downregulation of the MLL target Hoxa9 and impaired cell cycle progression. Structure-function analysis revealed two small eGFP-fused LEDGF/p75 peptides, LEDGF(424-435) and LEDGF(375-386) phenocopying these effects. Both LEDGF(325-530) and the smaller active peptides were able to disrupt the LEDGF/p75-MLL interaction. Expression of LEDGF(325-530) or LEDGF(375-386) fragments increased the latency period to disease development in vivo in a mouse bone marrow transplant model of MLL-AF9-induced AML. We conclude that small peptides disrupting the LEDGF/p75-MLL interface have selective anti-leukemic activity providing a direct rationale for the design of small molecule inhibitors targeting this interaction.

Alteration of cardiac progenitor cell potency in GRMD dogs.

Among the animal models of Duchenne muscular dystrophy (DMD), the Golden Retriever muscular dystrophy (GRMD) dog is considered the best model in terms of size and pathological onset of the disease. As in human patients presenting with DMD or Becker muscular dystrophies (BMD), the GRMD is related to a spontaneous X-linked mutation of dystrophin and is characterized by myocardial lesions. In this respect, GRMD is a useful model to explore cardiac pathogenesis and for the development of therapeutic protocols. To investigate whether cardiac progenitor cells (CPCs) isolated from healthy and GRMD dogs may differentiate into myocardial cell types and to test the feasibility of cell therapy for cardiomyopathies in a preclinical model of DMD, CPCs were isolated from cardiac biopsies of healthy and GRMD dogs. Gene profile analysis revealed an active cardiac transcription network in both healthy and GRMD CPCs. However, GRMD CPCs showed impaired self-renewal and cardiac differentiation. Population doubling and telomerase analyses highlighted earlier senescence and proliferation impairment in progenitors isolated from GRMD cardiac biopsies. Immunofluorescence analysis revealed that only wt CPCs showed efficient although not terminal cardiac differentiation, consistent with the upregulation of cardiac-specific proteins and microRNAs. Thus, the pathological condition adversely influences the cardiomyogenic differentiation potential of cardiac progenitors. Using PiggyBac transposon technology we marked CPCs for nuclear dsRed expression, providing a stable nonviral gene marking method for in vivo tracing of CPCs. Xenotransplantation experiments in neonatal immunodeficient mice revealed a valuable contribution of CPCs to cardiomyogenesis with homing differences between wt and dystrophic progenitors. These results suggest that cardiac degeneration in dystrophinopathies may account for the progressive exhaustion of local cardiac progenitors and shed light on cardiac stemness in physiological and pathological conditions. Furthermore, we provide essential information that canine CPCs may be used to alleviate cardiac involvement in a large preclinical model of DMD.

Efficient and stable transduction of dopaminergic neurons in rat substantia nigra by rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9.

Dysfunction of the nigrostriatal system is the major cause of Parkinson's disease (PD). This brain region is therefore an important target for gene delivery aiming at disease modeling and gene therapy. Recombinant adeno-associated viral (rAAV) vectors have been developed as efficient vehicles for gene transfer into the central nervous system. Recently, several serotypes have been described, with varying tropism for brain transduction. In light of the further development of a viral vector-mediated rat model for PD, we performed a comprehensive comparison of the transduction and tropism for dopaminergic neurons (DNs) in the adult Wistar rat substantia nigra (SN) of seven rAAV vector serotypes (rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9). All vectors were normalized by titer and volume, and stereotactically injected into the SN. Gene expression was assessed non-invasively and quantitatively in vivo by bioluminescence imaging at 2 and 5 weeks after injection, and was found to be stable over time. Immunohistochemistry at 6 weeks following injection revealed the most widespread enhanced green fluorescence protein expression and the highest number of positive nigral cells using rAAV 2/7, 2/9 and 2/1. The area transduced by rAAV 2/8 was smaller, but nevertheless almost equal numbers of nigral cells were targeted. Detailed confocal analysis revealed that serotype 2/7, 2/9, 2/1 and 2/8 transduced at least 70% of the DNs. In conclusion, these results show that various rAAV serotypes efficiently transduce nigral DNs, but significant differences in transgene expression pattern and level were observed.

Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors.

The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T(2)/T(2)(*) (spin-spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T(2)(*)-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging.

The fetal respiratory system as target for antenatal therapy.

The widespread use of prenatal ultrasound has made the fetus a patient. A number of conditions diagnosed as such may require therapy prior to birth. Herein we describe past, current and potential future procedures designed to treat pulmonary conditions in the antenatal period. When congenital cystic adenomatoid malformation (CCAM) is -associated with fetal hydrops, treatment is required. Prior to viability this may be in utero resection of the pathologic lung lobe or shunting of cystic lesions. More recently, fetuses with isolated congenital diaphragmatic hernia (CDH) with lethal lung hypoplasia have been offered percutaneous fetal tracheal occlusion to provoke lung growth. A very rare condition is laryngeal atresia, which requires peripartum re-establishment of the airways. As we get more -experience with access to the fetal airways, this may open the doors for novel therapies. One of these is gene delivery to treat fetuses with serious monogenic disorders or to induce transient overexpression of certain proteins. We review the individual hurdles that are being met by researchers when designing fetal gene therapeutic strategies, in particular for the fetal lung. Also the use of stem cells for pulmonary disorders is currently explored.

The fetal patient -- ethical aspects of fetal therapy.

The pregnant patient is a vulnerable subject, and even more so when a serious fetal condition is diagnosed. (Invasive) fetal therapy should only be offered when there is a good chance that the life of the fetus will be saved, or irreversible damage by the disease or disability is prevented. Following diagnosis of a potentially treatable condition, the patient needs to be referred to a center with sufficient expertise in diagnosis and all therapeutic options. Preferences of the physician towards one or another antenatal intervention is not at stake prior to that moment. When fetal therapy is justified--, it should be offered with full respect for maternal choice and individual assessment and perception of potential-- risks, and should be at the location where there is sufficient expertise. For therapies of unproven benefit, the absence of evidence must be disclosed, and therapy should only be undertaken with full voluntary consent of the mother. These ought to be undertaken within well designed and approved trials and only by experts in the treatment modality. Potential risks and eventual morbidities in case of therapeutic failure should be part of the counselling, neither-- should fetal therapy be presented as an alternative to termination of pregnancy.

On the cell biology of HIV integration from basic research to development of novel antiviral drugs.

Viruses in general, HIV in particular, use cellular proteins or cofactors to achieve their replication cycle. Our recent research efforts resulted in the identification and validation of two new cellular cofactors of HIV replication: LEDGF/p75 and transportin-SR2. LEDGF/p75 is a cellular transcriptional coactivator used by the virus to tether the preintegration complex onto the chromosome; transportin-SR2 is a cellular import factor used by the virus for nuclear import into the nucleus. After validation of LEDGF/p75 as an antiviral target, we initiated a drug discovery program to develop small molecule inhibitors of integrase-LEDGF/p75 interaction. Using molecular modeling, medicinal chemistry, crystallography and virology, our team developed LEDGINs, the first-in-class small molecule inhibitors of HIV targeting integrase/LEDGF/p75 interaction. As for transportin-SR2, this protein was identified by yeast-two-hybrid screening and validated as a nuclear import factor for HIV. A drug discovery program for inhibitors of nuclear import is ongoing. Together our research results provide a paradigm shift in antiviral research. Yes, small molecules can be developed inhibiting the protein-protein interaction between a viral protein and a cellular cofactor. In a broader perspective our research strongly supports the development of protein-protein interaction inhibitors as drugs.

Naturally occurring singleton residues in AAV capsid impact vector performance and illustrate structural constraints.

Vectors based on the adeno-associated virus (AAV) are attractive and versatile vehicles for in vivo gene transfer. The virus capsid is the primary interface with the cell that defines many pharmacological, immunological and molecular properties. Determinants of these interactions are often restricted to a limited number of capsid amino acids. In this study, a portfolio of novel AAV vectors was developed after a structure-function analysis of naturally occurring AAV capsid isolates. Singletons, which are particular residues on the AAV capsid that were variable in otherwise conserved amino acid positions, were found to impact on vector's ability to be manufactured or to transduce. Data for those residues that mapped to monomer-monomer interface regions on the particle structure suggested a role in particle assembly. The change of singleton residues to the conserved amino acid resulted in the rescue of many isolates that were defective on initial isolation. This led to the development of an AAV vector portfolio that encompasses six different clades and 3 other distinct AAV niches. Evaluation of the in vivo gene transfer efficiency of this portfolio after intravenous and intramuscular administration highlighted a clade-specific tropism. These studies further the design and selection of AAV capsids for gene therapy applications.

In vivo bioluminescence imaging in an experimental mouse model for dendritic cell based immunotherapy against malignant glioma.

The value of bioluminescence imaging (BLI) for experimental cancer models has become firmly established. We applied BLI to the GL261 glioma model in the context of dendritic cell (DC) immunotherapy. Initial validation revealed robust linear correlations between in vivo, ex vivo and in vitro luciferase activity measurements. Ex vivo BLI demonstrated midline crossing and leakage of tumor cells. Orthotopically challenged mice followed with BLI showed an initial adaptation phase, after which imaging data correlated linearly with stereologically determined tumor dimensions. Transition from healthy to moribund state corresponded with an increasing in vivo flux but the onset of neurological deficit was clearly delayed compared to the onset of in vivo flux increase. BLI was implemented in prophylactic immunotherapy and imaging data were prognostic for therapy outcome. Three distinct response patterns were detected. Our data underscore the feasibility of in vivo BLI in an experimental immunotherapeutic setting in the GL261 glioma model.

Design, Synthesis and antiHIV activity of Novel Isatine-Sulphonamides.

A series of novel isatine-sulphonamide derivatives have been synthesized by combining isatin derivatives with sulphonamides. The structure of the synthesized compounds were elucidated by spectral analysis (IR, NMR and Mass). Investigation of anti-HIV activity was done against HIV-1(IIIB) in MT-4 cells and HIV integrase inhibitory activity. 4-(1-acetyl-5-methyl-2-oxoindolin-3-ylideneamino)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide (SPIII-5ME-AC) inhibits the HIV Integrase enzymatic activity as both over all and strand transfer reaction and 4-(1-benzoyl-5-chloro-2-oxoindolin-3-ylideneamino)-N-(4,6-dimethylpyrimidin-2-yl)benzene sulfonamide (SPIII-5Cl-BZ) exhibits 36 percent maximum protection against HIV-1 at sub toxic concentration.

Selection of human immunodeficiency virus type 1 resistance against the pyranodipyrimidine V-165 points to a multimodal mechanism of action.

OBJECTIVES: We have previously identified the pyranodipyrimidines (PDPs) as a new class of integrase (IN) inhibitors. The most potent congener V-165 inhibits HIV-1 integration at low micromolar concentrations by inhibiting the binding of IN to the DNA. As part of pre-clinical studies with PDP, we wanted to investigate HIV resistance development against V-165 and to further characterize the physicochemical properties of the compound. METHODS: We selected PDP-resistant HIV-1 strains by growing the virus in the presence of increasing concentrations of V-165. The selected strains were analysed genotypically and phenotypically. Mutant IN enzymes were generated and evaluated in an enzymatic oligonucleotide-based assay for their activity and sensitivity to the different IN inhibitors. In addition, the antiviral effect of the compound on viral entry and integration was measured using quantitative PCR. RESULTS: Numerous mutations were detected in the RT, IN and env genes of the virus selected in the presence of V-165. Although V-165 inhibited integration in vivo as indicated by a decrease in the number of integrated proviruses, the compound also inhibited viral entry at a concentration of 19 microM. V-165 was poorly recovered from human hepatic microsomal matrix and 1% BSA. CONCLUSIONS: These data point to a multimodal mechanism of action. A quest for derivatives of V-165 that specifically target IN should be pursued.

Novel inhibitors of HIV-1 integration.

Human immunodeficiency virus (HIV) is the etiological agent of the acquired immune deficiency syndrome (AIDS). The current strategy for the treatment of HIV infection is called Highly Active Antiretroviral Therapy (HAART) and is based on cocktails of drugs that are currently approved by the Food and Drug Administration. These drugs include compounds that target the viral entry step and the enzymes reverse transcriptase or protease. The introduction of HAART has dramatically changed the landscape of HIV disease. Death from AIDS-related diseases has been reduced significantly since HAART came into use. Nevertheless it is not clear how long clinical benefit will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting other steps of the viral replication cycle may increase the potency of inhibition and delay resistance development. HIV integrase is an essential enzyme in the HIV life cycle and is an attractive target for new drug development. Despite years of intensive research, only two classes of compounds that inhibit integration have been identified until now, namely the diketo acids and the pyranodipyrimidines. In this review we will point to new potential antiviral targets related to retroviral integration that are amenable to drug development. We will describe the pitfalls of currently used integrase assays and propose new strategies and technologies for the discovery of HIV integration inhibitors. Furthermore, we will describe the two classes of integrase inhibitors and discuss their antiviral activity, molecular mechanism of anti-HIV action and the selection of HIV resistance against these drugs.

The integrase of the human immunodeficiency virus as a novel target for the antiviral therapy of AIDS.

Integration of the proviral DNA is a crucial step in the life cycle of the human immunodeficiency virus (HIV). Basic understanding of the cell biology of HIV integration will help the development of new antiviral drugs to complement the currently used cocktails of reverse transcriptase and protease inhibitors. Our research team has made major advancement in the field of antiviral research in recent years. The scientific-technological achievements were possible by the collaboration of a group of researchers with complementary research expertise. The integrase team is headed by Myriam Witvrouw and Zeger Debyser. After a general overview we summarized our most relevant publications. In the perspectives we referred to the ongoing work.

Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain.

HIV-derived lentiviral vectors are efficient vehicula to deliver genes into the brain and hold great promise for future gene therapy of neurodegenerative disorders. However, administration of the current vector preparations in mouse brain was found to induce a systemic immune response to vector proteins and a modest inflammation in the brain. Moreover, serum antibodies from vector-treated animals were capable of partially neutralizing lentiviral vector-mediated transduction in cell culture. To avoid this unexpected immune reaction, we have optimized new vector production and purification protocols. Purification by sucrose gradient ultracentrifugation abolished the immune response, but vector titers also decreased substantially. Lentiviral vector production in the absence of serum in the cell culture medium equally reduced immunogenicity without affecting transduction efficiency. These results have important implications for future clinical use of lentiviral vectors, and for the use of lentiviral vectors to create animal models for neurodegenerative diseases that have an important neuroinflammatory component.

HIV-1 integration as a target for antiretroviral therapy: a review.

Since the discovery of the human immunodeficiency virus type 1 (HIV-1) as the causative agent of AIDS in the early eighties, its spread has been dramatic. Current therapeutic strategies for the inhibition of viral replication employ a combination of drugs targeted at the viral reverse transcriptase and protease enzymes. The clinical benefit of this combination therapy is considerable, although often only transient, partly due to the emergence of multiple drug-resistant viral strains. The addition of new anti-HIV drugs targeting a third step of the viral replication may help in preventing resistance development. During HIV replication, the integration of the genome into the cellular chromosome is a vital step, which is catalysed by the viral integrase. The search for antiviral compounds capable of selective inhibition of integrase during viral replication is laborious and the large-scale screening programs for integrase inhibitors have thus far led to only one series of compounds that selectively inhibit the integration step of HIV replication, the diketo acids. In this review we summarize the current knowledge about HIV-1 integrase and integrase inhibitors. We address the issue why it is so difficult to find potent and selective integrase inhibitors, suitable to be included in a therapeutic drug combination and we propose new strategies for the discovery of integration inhibitors.

DNA-Dependent protein kinase is not required for efficient lentivirus integration.

How DNA is repaired after retrovirus integration is not well understood. DNA-dependent protein kinase (DNA-PK) is known to play a central role in the repair of double-stranded DNA breaks. Recently, a role for DNA-PK in retroviral DNA integration has been proposed (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Reduced transduction efficiency and increased cell death by apoptosis were observed upon retrovirus infection of cultured scid cells. We have used a human immunodeficiency virus (HIV) type 1 (HIV-1)-derived lentivirus vector system to further investigate the role of DNA-PK during integration. We measured lentivirus transduction of scid mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells. These cells are deficient in the catalytic subunit of DNA-PK and in Ku, the DNA-binding subunit of DNA-PK, respectively. At low vector titers, efficient and stable lentivirus transduction was obtained, excluding an essential role for DNA-PK in lentivirus integration. Likewise, the efficiency of transduction of HIV-derived vectors in scid mouse brain was as efficient as that in control mice, without evidence of apoptosis. We observed increased cell death in scid MEF and xrs-5 or xrs-6 cells, but only after transduction with high vector titers (multiplicity of infection [MOI], >1 transducing unit [TU]/cell) and subsequent passage of the transduced cells. At an MOI of <1 TU/cell, however, transduction efficiency was even higher in DNA-PK-deficient cells than in control cells. Taken together, the data suggest a protective role of DNA-PK against cellular toxicity induced by high levels of retrovirus integrase or integration. Another candidate cellular enzyme that has been claimed to play an important role during retrovirus integration is poly(ADP-ribose) polymerase (PARP). However, no inhibition of lentivirus vector-mediated transduction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, was observed. In conclusion, DNA-PK and PARP are not essential for lentivirus integration.

Polyanionic (i.e., polysulfonate) dendrimers can inhibit the replication of human immunodeficiency virus by interfering with both virus adsorption and later steps (reverse transcriptase/integrase) in the virus replicative cycle.

Polyanionic dendrimers were synthesized and evaluated for their antiviral effects. Phenyldicarboxylic acid (BRI6195) and naphthyldisulfonic acid (BRI2923) dendrimers were found to inhibit the replication of human immunodeficiency virus type 1 (HIV-1; strain III(B)) in MT-4 cells at a EC(50) of 0.1 and 0.3 microg/ml, respectively. The dendrimers were not toxic to MT-4 cells up to the highest concentrations tested (250 microg/ml). These compounds were also effective against various other HIV-1 strains, including clinical isolates, HIV-2 strains, simian immunodeficiency virus (SIV, strain MAC(251)), and HIV-1 strains that were resistant to reverse transcriptase inhibitors. HIV strains containing mutations in the envelope glycoprotein gp120 (engendering resistance to known adsorption inhibitors) displayed reduced sensitivity to the dendrimers. The compounds inhibited the binding of wild-type virus and recombinant virus (containing wild-type gp120) to MT-4 cells at concentrations comparable to those that inhibited the replication of HIV-1(III(B)) in these cells. Cellular uptake studies indicated that BRI2923, but not BRI6195, permeates into MT-4 and CEM cells. Accordingly, the naphtyldisulfonic acid dendrimer (BRI2923) proved able to inhibit later steps of the replication cycle of HIV, i.e., reverse transcriptase and integrase. NL4.3 strains resistant to BRI2923 were selected after passage of the virus in the presence of increasing concentrations of BRI2923. The virus mutants showed 15-fold reduced sensitivity to BRI2923 and cross-resistance to known adsorption inhibitors. However, these virus mutants were not cross-resistant to reverse transcriptase inhibitors or protease inhibitors. We identified several mutations in the envelope glycoprotein gp120 gene (i.e., V2, V3, and C3, V4, and C4 regions) of the BRI2923-resistant NL4.3 strains that were not present in the wild-type NL4.3 strain, whereas no mutations were found in the reverse transcriptase or integrase genes.

Viral entry as the primary target for the anti-HIV activity of chicoric acid and its tetra-acetyl esters.

The antiviral activity of L-chicoric acid against HIV-1 has been attributed previously to the inhibition of HIV-1 integration. This conclusion was based on the inhibition of integrase activity in enzymatic assays and the isolation of a resistant HIV strain with a mutation (G140S) in the integrase gene. Here we show that the primary antiviral target of L-CA and its analogs in cell culture is viral entry. L- and D-chicoric acid (L-CA and D-CA) and their respective tetra-acetyl esters inhibit the replication of HIV-1 (III(B) and NL4.3) and HIV-2 (ROD) in MT-4 cells at a 50% effective concentration (EC(50)) ranging from 1.7 to 70.6 microM. In a time-of-addition experiment, L-CA, D-CA, L-CATA, and D-CATA were found to interfere with an early event in the viral replication cycle. Moreover, L-CA, D-CA, and their analogs did not inhibit the replication of virus strains that were resistant toward polyanionic and polycationic compounds at subtoxic concentrations. Furthermore, HIV-1 strains resistant to L-CA and D-CA were selected in the presence of L-CA and D-CA, respectively. Mutations were found in the V2, V3, and V4 loop region of the envelope glycoprotein gp120 of the L-CA and D-CA-resistant NL4.3 strains that were not present in the wild-type NL4.3 strain. Recombination of the gp120 gene of the L-CA and D-CA resistant strain in a NL4.3 wild-type molecular clone fully rescued the phenotypic resistance toward L-CA and D-CA. No significant mutations were detected in the integrase gene of the drug-resistant virus strains. Although inhibition of HIV integrase activity by L-CA and its derivatives was confirmed in an oligonucleotide-driven assay, integrase carrying the G140S mutation was inhibited to the same extent as the wild-type integrase.

High-level expression of active HIV-1 integrase from a synthetic gene in human cells.

A synthetic gene encoding for HIV-1 integrase was designed to circumvent the intrinsic instability and the repressor elements present in the wild-type gene. High-level expression of HIV-1 integrase was obtained in various human cell lines independently of viral accessory proteins. A human 293T cell line was selected that stably expresses HIV-1 integrase and has growth kinetics comparable to the parental cell line. The enzyme was localized in the nucleus and remained stably associated with the chromosomes during mitosis. Lentiviral vector particles carrying the inactivating D64V mutation in the integrase gene were capable of stably transducing 293T cells when complemented in the producer cells with integrase expressed from the synthetic gene. When the cell line that stably expresses integrase was infected with the defective viral particles, complementation of integrase activity was detected as well. Expression of active HIV-1 integrase in human cells will facilitate the study of the interplay between host and viral factors during integration.