Chemical, Biochemical, Cellular, and Physiological Characterization of Leucettinib-21, a Down Syndrome and Alzheimer's Disease Drug Candidate.

Leucettinibs are substituted 2-aminoimidazolin-4-ones (inspired by the marine sponge natural product Leucettamine B) developed as pharmacological inhibitors of DYRK1A (dual-specificity, tyrosine phosphorylation-regulated kinase 1A), a therapeutic target for indications such as Down syndrome and Alzheimer's disease. Leucettinib-21 was selected as a drug candidate following extensive structure/activity studies and multiparametric evaluations. We here report its physicochemical properties (X-ray powder diffraction, differential scanning calorimetry, stability, solubility, crystal structure) and drug-like profile. Leucettinib-21's selectivity (analyzed by radiometric, fluorescence, interaction, thermal shift, residence time assays) reveals DYRK1A as the first target but also some "off-targets" which may contribute to the drug's biological effects. Leucettinib-21 was cocrystallized with CLK1 and modeled in the DYRK1A structure. Leucettinib-21 inhibits DYRK1A in cells (demonstrated by direct catalytic activity and phosphorylation levels of Thr286-cyclin D1 or Thr212-Tau). Leucettinib-21 corrects memory disorders in the Down syndrome mouse model Ts65Dn and is now entering safety/tolerance phase 1 clinical trials.

Leucettinibs, a Class of DYRK/CLK Kinase Inhibitors Inspired by the Marine Sponge Natural Product Leucettamine B.

Dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) recently attracted attention due to their central involvement in various pathologies. We here describe a family of DYRK/CLK inhibitors derived from Leucettines and the marine natural product Leucettamine B. Forty-five N2-functionalized 2-aminoimidazolin-4-ones bearing a fused [6 + 5]-heteroarylmethylene were synthesized. Benzothiazol-6-ylmethylene was selected as the most potent residue among 15 different heteroarylmethylenes. 186 N2-substituted 2-aminoimidazolin-4-ones bearing a benzothiazol-6-ylmethylene, collectively named Leucettinibs, were synthesized and extensively characterized. Subnanomolar IC50 (0.5-20 nM on DYRK1A) inhibitors were identified and one Leucettinib was modeled in DYRK1A and co-crystallized with CLK1 and the weaker inhibited off-target CSNK2A1. Kinase-inactive isomers of Leucettinibs (>3-10 µM on DYRK1A), named iso-Leucettinibs, were synthesized and characterized as suitable negative control compounds for functional experiments. Leucettinibs, but not iso-Leucettinibs, inhibit the phosphorylation of DYRK1A substrates in cells. Leucettinibs provide new research tools and potential leads for further optimization toward therapeutic drug candidates.

Comparative Efficacy and Selectivity of Pharmacological Inhibitors of DYRK and CLK Protein Kinases.

Dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) play a large variety of cellular functions and are involved in several diseases (cognitive disorders, diabetes, cancers, etc.). There is, thus, growing interest in pharmacological inhibitors as chemical probes and potential drug candidates. This study presents an unbiased evaluation of the kinase inhibitory activity of a library of 56 reported DYRK/CLK inhibitors on the basis of comparative, side-by-side, catalytic activity assays on a panel of 12 recombinant human kinases, enzyme kinetics (residence time and Kd), in-cell inhibition of Thr-212-Tau phosphorylation, and cytotoxicity. The 26 most active inhibitors were modeled in the crystal structure of DYRK1A. The results show a rather large diversity of potencies and selectivities among the reported inhibitors and emphasize the difficulties to avoid "off-targets" in this area of the kinome. The use of a panel of DYRKs/CLKs inhibitors is suggested to analyze the functions of these kinases in cellular processes.

Epigenetic therapy to enhance therapeutic effects of PD-1 inhibition in therapy-resistant melanoma.

Targeted therapy and immunotherapy have revolutionized the treatment of metastatic skin melanoma but around half of all patients develop resistance early or late during treatment. The situation is even worse for patients with metastatic uveal melanoma (UM). Here we hypothesized that the immunotherapy of therapy-resistant skin melanoma or UM can be enhanced by epigenetic inhibitors. Cultured B16F10 cells and human UM cells were treated with the histone deacetylase inhibitor (HDACi) entinostat or BETi JQ1. Entinostat-induced HLA expression and PD-L1, but JQ1 did not. A syngeneic mouse model carrying B16-F10 melanoma cells was treated with PD-1 and CTLA4 inhibitors, which was curative. Co-treatment with the bioavailable BETi iBET726 impaired the immunotherapy effect. Monotherapy of a B16-F10 mouse model with anti-PD-1 resulted in a moderate therapeutic effect that could be enhanced by entinostat. Mice carrying PD-L1 knockout B16-F10 cells were also sensitive to entinostat. This suggests HDAC inhibition and immunotherapy could work in concert. Indeed, co-cultures of UM with HLA-matched melanoma-specific tumor-infiltrating lymphocytes (TILs) resulted in higher TIL-mediated melanoma killing when entinostat was added. Further exploration of combined immunotherapy and epigenetic therapy in metastatic melanoma resistant to PD-1 inhibition is warranted.

Structure-Activity Relationship in the Leucettine Family of Kinase Inhibitors.

The protein kinase DYRK1A is involved in Alzheimer's disease, Down syndrome, diabetes, viral infections, and leukemia. Leucettines, a family of 2-aminoimidazolin-4-ones derived from the marine sponge alkaloid Leucettamine B, have been developed as pharmacological inhibitors of DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). We report here on the synthesis and structure-activity relationship (SAR) of 68 Leucettines. Leucettines were tested on 11 purified kinases and in 5 cellular assays: (1) CLK1 pre-mRNA splicing, (2) Threonine-212-Tau phosphorylation, (3) glutamate-induced cell death, (4) autophagy and (5) antagonism of ligand-activated cannabinoid receptor CB1. The Leucettine SAR observed for DYRK1A is essentially identical for CLK1, CLK4, DYRK1B, and DYRK2. DYRK3 and CLK3 are less sensitive to Leucettines. In contrast, the cellular SAR highlights correlations between inhibition of specific kinase targets and some but not all cellular effects. Leucettines deserve further development as potential therapeutics against various diseases on the basis of their molecular targets and cellular effects.

Dual-Specificity, Tyrosine Phosphorylation-Regulated Kinases (DYRKs) and cdc2-Like Kinases (CLKs) in Human Disease, an Overview.

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRK1A, 1B, 2-4) and cdc2-like kinases (CLK1-4) belong to the CMGC group of serine/threonine kinases. These protein kinases are involved in multiple cellular functions, including intracellular signaling, mRNA splicing, chromatin transcription, DNA damage repair, cell survival, cell cycle control, differentiation, homocysteine/methionine/folate regulation, body temperature regulation, endocytosis, neuronal development, synaptic plasticity, etc. Abnormal expression and/or activity of some of these kinases, DYRK1A in particular, is seen in many human nervous system diseases, such as cognitive deficits associated with Down syndrome, Alzheimer's disease and related diseases, tauopathies, dementia, Pick's disease, Parkinson's disease and other neurodegenerative diseases, Phelan-McDermid syndrome, autism, and CDKL5 deficiency disorder. DYRKs and CLKs are also involved in diabetes, abnormal folate/methionine metabolism, osteoarthritis, several solid cancers (glioblastoma, breast, and pancreatic cancers) and leukemias (acute lymphoblastic leukemia, acute megakaryoblastic leukemia), viral infections (influenza, HIV-1, HCMV, HCV, CMV, HPV), as well as infections caused by unicellular parasites (Leishmania, Trypanosoma, Plasmodium). This variety of pathological implications calls for (1) a better understanding of the regulations and substrates of DYRKs and CLKs and (2) the development of potent and selective inhibitors of these kinases and their evaluation as therapeutic drugs. This article briefly reviews the current knowledge about DYRK/CLK kinases and their implications in human disease.

Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells.

Chk1 kinase is downstream of the ATR kinase in the sensing of improper replication. Previous cell culture studies have demonstrated that Chk1 is essential for replication. Indeed, Chk1 inhibitors are efficacious against tumors with high-level replication stress such as Myc-induced lymphoma cells. Treatment with Chk1 inhibitors also combines well with certain chemotherapeutic drugs, and effects associate with the induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function have relied on the use of inhibitors. Whether or not a mouse or cancer cells could survive if a kinase-dead form of Chk1 is expressed has not been investigated before. Here, we generate a mouse model that expresses a kinase-dead (D130A) allele in the mouse germ line. We find that this mouse is overtly normal and does not have problems with erythropoiesis with aging as previously been shown for a mouse expressing one null allele. However, similar to a null allele, homozygous kinase-dead mice cannot be generated, and timed pregnancies of heterozygous mice suggest lethality of homozygous blastocysts at around the time of implantation. By breeding the kinase-dead Chk1 mouse with a conditional allele, we are able to demonstrate that expression of only one kinase-dead allele, but no wild-type allele, of Chek1 is lethal for Myc-induced cancer cells. Finally, treatment of melanoma cells with tumor-infiltrating T cells or CAR-T cells is effective even if Chk1 is inhibited, suggesting that Chk1 inhibitors can be safely administered in patients where immunotherapy is an essential component of the arsenal against cancer.

Correction: A patient-derived xenograft pre-clinical trial reveals treatment responses and a resistance mechanism to karonudib in metastatic melanoma.

On Pubmed, the name of co-author Roger Olofsson Bagge appeared incorrectly as "Bagge RO" instead of "Olofsson Bagge, Roger". This has been corrected in the PDF and HTML versions.

Biological Characterization of 8-Cyclopropyl-2-(pyridin-3-yl)thiazolo[5,4-f]quinazolin-9(8H)-one, a Promising Inhibitor of DYRK1A.

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) hyperactivity has been linked to the development of a number of human malignancies. DYRK1A is the most studied family member, and the discovery of novel specific inhibitors is attracting considerable interest. The 8-cyclopropyl-2(pyridin-3-yl)thiazolo[5,4-f]quinazolin-9(8H)-one (also called FC162) was found to be a promising inhibitor of DYRK1A and was characterized in biological experiments, by western transfer and flow cytometry on SH-SY5Y and pre-B cells. Here, the results obtained with FC162 are compared to well-characterized known DYRK1A inhibitors (e.g., Leucettine L41 and EHT1610).

Novel Mouse Tauopathy Model for Repetitive Mild Traumatic Brain Injury: Evaluation of Long-Term Effects on Cognition and Biomarker Levels After Therapeutic Inhibition of Tau Phosphorylation.

Traumatic brain injury (TBI) is a risk factor for a group of neurodegenerative diseases termed tauopathies, which includes Alzheimer's disease and chronic traumatic encephalopathy (CTE). Although TBI is stratified by impact severity as either mild (m), moderate or severe, mTBI is the most common and the most difficult to diagnose. Tauopathies are pathologically related by the accumulation of hyperphosphorylated tau (P-tau) and increased total tau (T-tau). Here we describe: (i) a novel human tau-expressing transgenic mouse model, TghTau/PS1, to study repetitive mild closed head injury (rmCHI), (ii) quantitative comparison of T-tau and P-tau from brain and plasma in TghTau/PS1 mice over a 12 month period following rmCHI (and sham), (iii) the usefulness of P-tau as an early- and late-stage blood-based biochemical biomarker for rmCHI, (iii) the influence of kinase-targeted therapeutic intervention on rmCHI-associated cognitive deficits using a combination of lithium chloride (LiCl) and R-roscovitine (ros), and (iv) correlation of behavioral and cognitive changes with concentrations of the brain and blood-based T-tau and P-tau. Compared to sham-treated mice, behavior changes and cognitive deficits of rmCHI-treated TghTau/PS1 mice correlated with increases in both cortex and plasma T-tau and P-tau levels over 12 months. In addition, T-tau, but more predominantly P-tau, levels were significantly reduced in the cortex and plasma by LiCl + ros approaching the biomarker levels in sham and drug-treated sham mice (the drugs had only modest effects on the T-tau and P-tau levels in sham mice) throughout the 12 month study period. Furthermore, although we also observed a reversal of the abnormal behavior and cognitive deficits in the drug-treated rmCHI mice (compared to the untreated rmCHI mice) throughout the time course, these drug-treated effects were most pronounced up until 10 and 12 months where the abnormal behavior and cognition deficits began to gradually increase. These studies describe: (a) a translational relevant animal model for TBI-linked tauopathies, and (b) utilization of T-tau and P-tau as rmCHI biomarkers in plasma to monitor novel therapeutic strategies and treatment regimens for these neurodegenerative diseases.

HER2 CAR-T Cells Eradicate Uveal Melanoma and T-cell Therapy-Resistant Human Melanoma in IL2 Transgenic NOD/SCID IL2 Receptor Knockout Mice.

Chimeric antigen receptors (CAR) can transmit signals akin to those from activated T-cell receptors when bound to a cell surface target. CAR-expressing T cells against CD19 can cause curative effects in leukemia and lymphoma and is approved for clinical use. However, no CAR-T therapy is currently approved for use in solid tumors. We hypothesize that the resistance of solid tumors to CAR-T can be overcome by similar means as those used to reactivate tumor-infiltrating T lymphocytes (TIL), for example, by cytokines or immune checkpoint blockade. Here we demonstrate that CAR-T cells directed against HER2 can kill uveal and cutaneous melanoma cells in vitro and in vivo. Curative effects in vivo were only observed in xenografts grown in a NOD/SCID IL2 receptor gamma (NOG) knockout mouse strain transgenic for human IL2. The effect was target-specific, as CRISPR/Cas9-mediated disruption of HER2 in the melanoma cells abrogated the killing effect of the CAR-T cells. The CAR-T cells were also able to kill melanoma cells from patients resistant to adoptive T-cell transfer (ACT) of autologous TILs. Thus, CAR-T therapy represents an option for patients that do not respond to immunotherapy with ACT of TIL or immune checkpoint blockade. In addition, our data highlight the use of IL2 transgenic NOG mice as models to prove efficacy of CAR-T-cell products, possibly even in a personalized manner. SIGNIFICANCE: These findings demonstrate that a novel humanized mouse model can help clinical translation of CAR-T cells against uveal and cutaneous melanoma that do not respond to TIL therapy or immune checkpoint blockade.

A patient-derived xenograft pre-clinical trial reveals treatment responses and a resistance mechanism to karonudib in metastatic melanoma.

Karonudib (TH1579) is a novel compound that exerts anti-tumor activities and has recently entered phase I clinical testing. The aim of this study was to conduct a pre-clinical trial in patient-derived xenografts to identify the possible biomarkers of response or resistance that could guide inclusion of patients suffering from metastatic melanoma in phase II clinical trials. Patient-derived xenografts from 31 melanoma patients with metastatic disease were treated with karonudib or a vehicle for 18 days. Treatment responses were followed by measuring tumor sizes, and the models were categorized in the response groups. Tumors were harvested and processed for RNA sequencing and protein analysis. To investigate the effect of karonudib on T-cell-mediated anti-tumor activities, tumor-infiltrating T cells were injected in mice carrying autologous tumors and the mice treated with karonudib. We show that karonudib has heterogeneous anti-tumor effect on metastatic melanoma. Thus, based on the treatment responses, we could divide the 31 patient-derived xenografts in three treatment groups: progression group (32%), suppression group (42%), and regression group (26%). Furthermore, we show that karonudib has anti-tumor effect, irrespective of major melanoma driver mutations. Also, we identify high expression of ABCB1, which codes for p-gp pumps as a resistance biomarker. Finally, we show that karonudib treatment does not hamper T-cell-mediated anti-tumor responses. These findings can be used to guide future use of karonudib in clinical use with a potential approach as precision medicine.

Mutational Signature and Transcriptomic Classification Analyses as the Decisive Diagnostic Tools for a Cancer of Unknown Primary.

PURPOSE: Cancer of unknown primary is a group of metastatic tumors in which the standard diagnostic workup fails to identify the site of origin of the tumor. The potential impact of precision oncology on this group of patients is large, because actionable driver mutations and a correct diagnosis could provide treatment options otherwise not available for patients with these fatal cancers. This study investigated if comprehensive genomic analyses could provide information on the origin of the tumor. PATIENTS AND METHODS: Here we describe a patient whose tumor was misdiagnosed at least three times. Next-generation sequencing, a patient-derived xenograft mouse model, and bioinformatics were used to identify an actionable mutation, predict resistance development to the targeted therapy, and correctly diagnose the origin of the tumor. Transcriptomic classification was benchmarked using The Cancer Genome Atlas (TCGA). RESULTS: Despite the lack of a known primary tumor site and the absence of diagnostic immunohistochemical markers, the origin of the patient's tumor was established using the novel bioinformatic workflow. This included a mutational signature analysis of the sequenced metastases and comparison of their transcriptomic profiles to a pan-cancer panel of tumors from TCGA. We further discuss the strengths and limitations of the latter approaches in the context of three potentially incorrectly diagnosed TCGA lung tumors. CONCLUSION: Comprehensive genomic analyses can provide information on the origin of tumors in patients with cancer of unknown primary.

BET bromodomain inhibitors synergize with ATR inhibitors in melanoma.

This corrects the article DOI: 10.1038/cddis.2017.383.

Clinical responses to adoptive T-cell transfer can be modeled in an autologous immune-humanized mouse model.

Immune checkpoint inhibitors and adoptive cell transfer (ACT) of autologous tumor-infiltrating T cells have shown durable responses in patients with melanoma. To study ACT and immunotherapies in a humanized model, we have developed PDXv2.0 - a melanoma PDX model where tumor cells and tumor-infiltrating T cells from the same patient are transplanted sequentially in non-obese diabetic/severe combined immune-deficient/common gamma chain (NOG/NSG) knockout mouse. Key to T-cell survival/effect in this model is the continuous presence of interleukin-2 (IL-2). Tumors that grow in PDXv2.0 are eradicated if the autologous tumor cells and T cells come from a patient that exhibited an objective response to ACT in the clinic. However, T cells from patients that are non-responders to ACT cannot kill tumor cells in PDXv2.0. Taken together, PDXv2.0 provides the potential framework to further model genetically diverse human cancers for assessing the efficacy of immunotherapies as well as combination therapies.Combining different types of immune therapies might benefit certain patients. Here, the authors develop an autologous immune-humanized melanoma mouse model that allows the preclinical assessment of cancer cell-T cell interactions from each individual patient and the benefits of immunotherapies combinations.

BET bromodomain inhibitors synergize with ATR inhibitors in melanoma.

Metastatic malignant melanoma continues to be a challenging disease despite clinical translation of the comprehensive understanding of driver mutations and how melanoma cells evade immune attack. In Myc-driven lymphoma, efficacy of epigenetic inhibitors of the bromodomain and extra-terminal domain (BET) family of bromodomain proteins can be enhanced by combination therapy with inhibitors of the DNA damage response kinase ATR. Whether this combination is active in solid malignancies like melanoma, and how it relates to immune therapy, has not previously investigated. To test efficacy and molecular consequences of combination therapies cultured melanoma cells were used. To assess tumor responses to therapies in vivo we use patient-derived xenografts and B6 mice transplanted with B16F10 melanoma cells. Concomitant inhibition of BET proteins and ATR of cultured melanoma cells resulted in similar effects as recently shown in lymphoma, such as induction of apoptosis and p62, implicated in autophagy, senescence-associated secretory pathway and ER stress. In vivo, apoptosis and suppression of subcutaneous growth of patient-derived melanoma and B16F10 cells were observed. Our data suggest that ATRI/BETI combination therapies are effective in melanoma.

Acquired Immune Resistance Follows Complete Tumor Regression without Loss of Target Antigens or IFNγ Signaling.

Cancer immunotherapy can result in durable tumor regressions in some patients. However, patients who initially respond often experience tumor progression. Here, we report mechanistic evidence of tumoral immune escape in an exemplary clinical case: a patient with metastatic melanoma who developed disease recurrence following an initial, unequivocal radiologic complete regression after T-cell-based immunotherapy. Functional cytotoxic T-cell responses, including responses to one mutant neoantigen, were amplified effectively with therapy and generated durable immunologic memory. However, these immune responses, including apparently effective surveillance of the tumor mutanome, did not prevent recurrence. Alterations of the MHC class I antigen-processing and presentation machinery (APM) in resistant cancer cells, but not antigen loss or impaired IFNγ signaling, led to impaired recognition by tumor-specific CD8+ T cells. Our results suggest that future immunotherapy combinations should take into account targeting cancer cells with intact and impaired MHC class I-related APM. Loss of target antigens or impaired IFNγ signaling does not appear to be mandatory for tumor relapse after a complete radiologic regression. Personalized studies to uncover mechanisms leading to disease recurrence within each individual patient are warranted. Cancer Res; 77(17); 4562-6. ©2017 AACR.

A retro-inverso cell-penetrating peptide for siRNA delivery.

BACKGROUND: Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases. RESULTS: Here, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression. CONCLUSIONS: This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins). Graphical abstract RICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down.

Optimisation of vectorisation property: A comparative study for a secondary amphipathic peptide.

RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation and intracellular delivery. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. In this context, the secondary amphipathic peptide CADY has shown to form stable siRNA complexes and to improve their cellular uptake independent of the endosomal pathway. In the present work, we have described the parameters influencing CADY nanoparticle formation (buffers, excipients, presence of serum, etc.), and have followed in details the CPP:siRNA self-assembly. Once optimal conditions were determined, we have compared the ability of seven different CADY analogues to form siRNA-loaded nanoparticles compared to CADY:siRNA. First of all, we were able to show by biophysical methods that structural polymorphism (α-helix) is an important prerequisite for stable nanoparticle formation independently of occurring sequence mutations. Luciferase assays revealed that siRNA complexed to CADY-K (shorter version) shows better knock-down efficiency on Neuro2a-Luc(+) and B16-F10-Luc(+) cells compared to CADY:siRNA. Altogether, CADY-K is an ideal candidate for further application especially with regards to ex vivo or in vivo applications.

Efficient in vivo transfection and safety profile of a CpG-free and codon optimized luciferase plasmid using a cationic lipophosphoramidate in a multiple intravenous administration procedure.

As any drug, the success of gene therapy is largely dependent on the vehicle that has to selectively and efficiently deliver therapeutic nucleic acids into targeted cells with minimal side-effects. In the case of chronic diseases that require a life-long treatment, non-viral gene delivery vehicles are less likely to induce an immune response, thereby allowing for repeated administration. Beyond the gene delivery efficiency of a given vector, the nature of nucleic acid constructs also has a central importance in gene therapy protocols. Herein, we investigated the impact of two firefly luciferase encoding plasmids on the transgene expression profile following systemic delivery of lipoplexes in mice, as well as their potential to be safely and efficiently readministered. Whereas pTG11033 plasmid is driven by a strong ubiquitous cytomegalovirus promoter, pGM144 plasmid, which has been designed to avoid inflammation and provide sustained transgene expression in lungs, is CpG-free and is under control of the human elongation factor-1 alpha promoter. Combined to the efficient cationic lipophosphoramidate BSV4, bioluminescence data showed that both plasmids were mostly expressed in the lungs of mice following a primary injection of lipoplexes. However, mice transfected with pGM144 exhibited a higher and more sustained transgene expression than those treated with pTG11033. Repeated administration studies revealed that several injections of lipoplexes could lead to similar transgene expression profiles if an interval of several weeks between subsequent injections was respected. A transient hepatotoxicity and a partial inflammatory response were caused by lipoplex injection, irrespective of the plasmid used. Altogether, these results indicate that repeated systemic administration of lipophosphoramidate-based lipoplexes in mice conducts to an effective lung transfection without serious side effects, and highlight the need to use long-lasting expressing and well tolerated plasmids in order to efficiently renew transgene expression by the successive doses.