Retroviral Gene Therapy: May The Fibronectin Be With You

poster abstractReplication incompetent retroviral vectors are currently used in phase 1 clinical trials for genetic therapy of disorders of the blood and the immune system, as vector integration into the genome of target stem cells provides stable long-term expression of the therapeutic transgene. We have previously shown that co-localization of the viral particles and the target cells on the recombinant fibronectin fragment CH-296 enhances the retroviral gene transfer efficiency into primitive hematopoietic cells including stem cells. Here, we report additional technical details for improving the gene transfer efficiencies into hematopoietic cell lines, primary human T-cells and CD34+ cells and demonstrate that CH-296 can be used at least three times without any loss of efficiency. Finally, we expand the range of viral proteins known to directly bind to fibronectin CH-296 to the commonly used VSV-G, GaLV and foamyviral (FV) envelope

Therapeutic Strategies and Biomarkers to Modulate PARP Activity for Targeted Cancer Therapy

Poly-(ADP-ribose) polymerase 1 (PARP1) is commonly known for its vital role in DNA damage response and repair. However, its enzymatic activity has been linked to a plethora of physiological and pathophysiological transactions ranging from cellular proliferation, survival and death. For instance, malignancies with BRCA1/2 mutations heavily rely on PARP activity for survival. Thus, the use of PARP inhibitors is a well-established intervention in these types of tumors. However, recent studies indicate that the therapeutic potential of attenuating PARP1 activity in recalcitrant tumors, especially where PARP1 is aberrantly overexpressed and hyperactivated, may extend its therapeutic utility in wider cancer types beyond BRCA-deficiency. Here, we discuss treatment strategies to expand the tumor-selective therapeutic application of PARP inhibitors and novel approaches with predictive biomarkers to perturb NAD+ levels and hyperPARylation that inactivate PARP in recalcitrant tumors. We also provide an overview of genetic alterations that transform non-BRCA mutant cancers to a state of “BRCAness” as potential biomarkers for synthetic lethality with PARP inhibitors. Finally, we discuss a paradigm shift for the use of novel PARP inhibitors outside of cancer treatment, where it has the potential to rescue normal cells from severe oxidative damage during ischemia-reperfusion injury induced by surgery and radiotherapy

Systemic Injection of RPE65-Programmed Bone Marrow-Derived Cells Prevents Progression of Chronic Retinal Degeneration

Bone marrow stem and progenitor cells can differentiate into a range of non-hematopoietic cell types, including retinal pigment epithelium (RPE)-like cells. In this study, we programmed bone marrow-derived cells (BMDCs) ex vivo by inserting a stable RPE65 transgene using a lentiviral vector. We tested the efficacy of systemically administered RPE65-programmed BMDCs to prevent visual loss in the superoxide dismutase 2 knockdown (Sod2 KD) mouse model of age-related macular degeneration. Here, we present evidence that these RPE65-programmed BMDCs are recruited to the subretinal space, where they repopulate the RPE layer, preserve the photoreceptor layer, retain the thickness of the neural retina, reduce lipofuscin granule formation, and suppress microgliosis. Importantly, electroretinography and optokinetic response tests confirmed that visual function was significantly improved. Mice treated with non-modified BMDCs or BMDCs pre-programmed with LacZ did not exhibit significant improvement in visual deficit. RPE65-BMDC administration was most effective in early disease, when visual function and retinal morphology returned to near normal, and less effective in late-stage disease. This experimental paradigm offers a minimally invasive cellular therapy that can be given systemically overcoming the need for invasive ocular surgery and offering the potential to arrest progression in early AMD and other RPE-based diseases

Improving the Transduction of Bone Marrow-Derived Cells with an Integrase-Defective Lentiviral Vector

In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow-derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is concluded that IDLV3-RPE65 is appropriate for programming BMDCs to RPE-like cells

NQO1-Bioactivatable Therapeutics as Radiosensitizers for Cancer Treatment

Developing cancer therapeutics that radiosensitize in a tumor-selective manner remains an ideal. We developed a novel means of radiosensitization, exploiting NAD(P)H:Quinone Oxidoreductase 1 (NQO1) overexpression, and lowered catalase expression in solid human tumors using NQO1-bioactivatable drugs. Non-small cell lung (NSCLC), pancreatic (PDAC), prostate, and breast cancers overexpress NQO1. Ionizing radiation (IR) creates a spectrum of DNA lesions, including lethal DNA double-strand breaks (DSBs), and mutagenic but rarely lethal altered DNA bases and DNA single-strand breaks (SSBs). NQO1-bioactivatable drugs (e.g., β-lapachone and deoxynyboquiones) also promote abasic DNA lesions and SSBs. These hyperactivate poly (ADP-ribose) polymerase 1 (PARP1) and dramatically increase calcium release from the endoplasm reticulum (ER). Exposure of human cancer cells overexpressing NQO1 to NQO1-bioactivatable drugs immediately following IR, therefore, hyperactivates PARP1 synergistically, which in turn depletes NAD+ and ATP, inhibiting DSB repair. Ultimately, this leads to cell death. Combining IR with NQO1-bioactivatable drugs allows for a reduction in drug dose. Similarly, a lower IR dose can be used in combination with the drug, reducing the effects of IR on normal tissue. The combination treatment is effective in preclinical animal models with NSCLC, prostate, and head and neck xenografts, indicating that clinical trials are warranted

Targeting Base Excision Repair in Cancer: NQO1-Bioactivatable Drugs Improve Tumor Selectivity and Reduce Treatment Toxicity Through Radiosensitization of Human Cancer

Ionizing radiation (IR) creates lethal DNA damage that can effectively kill tumor cells. However, the high dose required for a therapeutic outcome also damages healthy tissue. Thus, a therapeutic strategy with predictive biomarkers to enhance the beneficial effects of IR allowing a dose reduction without losing efficacy is highly desirable. NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in the majority of recalcitrant solid tumors in comparison with normal tissue. Studies have shown that NQO1 can bioactivate certain quinone molecules (e.g., ortho-naphthoquinone and β-lapachone) to induce a futile redox cycle leading to the formation of oxidative DNA damage, hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1), and catastrophic depletion of NAD+ and ATP, which culminates in cellular lethality via NAD+-Keresis. However, NQO1-bioactivatable drugs induce methemoglobinemia and hemolytic anemia at high doses. To circumvent this, NQO1-bioactivatable agents have been shown to synergize with PARP1 inhibitors, pyrimidine radiosensitizers, and IR. This therapeutic strategy allows for a reduction in the dose of the combined agents to decrease unwanted side effects by increasing tumor selectivity. In this review, we discuss the mechanisms of radiosensitization between NQO1-bioactivatable drugs and IR with a focus on the involvement of base excision repair (BER). This combination therapeutic strategy presents a unique tumor-selective and minimally toxic approach for targeting solid tumors that overexpress NQO1

Endotoxin Preconditioning Reprograms S1 Tubules and Macrophages to Protect the Kidney

Preconditioning with a low dose of endotoxin confers unparalleled protection against otherwise lethal models of sepsis. The mechanisms of preconditioning have been investigated extensively in isolated immune cells such as macrophages. However, the role of tissue in mediating the protective response generated by preconditioning remains unknown. Here, using the kidney as a model organ, we investigated cell type-specific responses to preconditioning. Compared with preadministration of vehicle, endotoxin preconditioning in the cecal ligation and puncture mouse model of sepsis led to significantly enhanced survival and reduced bacterial load in several organs. Furthermore, endotoxin preconditioning reduced serum levels of proinflammatory cytokines, upregulated molecular pathways involved in phagocytosis, and prevented the renal function decline and injury induced in mice by a toxic dose of endotoxin. The protective phenotype involved the clustering of macrophages around S1 segments of proximal tubules, and full renal protection required both macrophages and renal tubular cells. Using unbiased S1 transcriptomic and tissue metabolomic approaches, we identified multiple protective molecules that were operative in preconditioned animals, including molecules involved in antibacterial defense, redox balance, and tissue healing. We conclude that preconditioning reprograms macrophages and tubules to generate a protective environment, in which tissue health is preserved and immunity is controlled yet effective. Endotoxin preconditioning can thus be used as a discovery platform, and understanding the role and participation of both tissue and macrophages will help refine targeted therapies for sepsis

Lack-of P-limitation of phytoplankton and heterotrophic prokaryotes in surface waters of three anticyclonic eddies in the stratified Mediterranean Sea

International audienceWe investigated the identity of the limiting nutrient of the pelagic microbial food web in the Mediter-ranean Sea using nutrient manipulated microcosms during summer 2008. Experiments were carried out with surface waters at the center of anticyclonic eddies in the Western Basin, the Ionian Basin, and the Levantine Basin. In situ, the ratio of N to P was always higher in both dissolved and particulate organic fractions compared to the Redfield ratio , suggesting a relative P-starvation. In each experiment, four different treatments in triplicates (addition of ammo-nium, phosphate, a combination of both, and the unamended control) were employed and chemical and biological parameters monitored throughout a 3-4 day incubation. Temporal changes of turnover time of phosphate and ATP, and alkaline phosphatase activity during the incubation suggested that the phytoplankton and heterotrophic prokaryotes (Hprok) communities were not P-limited at the sites. Furthermore, Correspondence to: T. Tanaka ([email protected]) statistical comparison among treatments at the end of the incubation did not support a hypothesis of P-limitation at the three study sites. In contrast, primary production was consistently limited by N, and Hprok growth was not limited by N nor P in the Western Basin, but N-limited in the Ionian Basin, and N and P co-limited in the Levantine Basin. Our results demonstrated the gap between biogeochemical features (an apparent P-starved status) and biological responses (no apparent P-limitation). We question the general notion that Mediterranean surface waters are limited by P alone during the stratified period

The archaeal Dps nanocage targets kidney proximal tubules via glomerular filtration

Nature exploits cage-like proteins for a variety of biological purposes, from molecular packaging and cargo delivery to catalysis. These cage-like proteins are of immense importance in nanomedicine due to their propensity to self-assemble from simple identical building blocks to highly ordered architecture and the design flexibility afforded by protein engineering. However, delivery of protein nanocages to the renal tubules remains a major challenge because of the glomerular filtration barrier, which effectively excludes conventional size nanocages. Here, we show that DNA-binding protein from starved cells (Dps) — the extremely small archaeal antioxidant nanocage — is able to cross the glomerular filtration barrier and is endocytosed by the renal proximal tubules. Using a model of endotoxemia, we present an example of the way in which proximal tubule–selective Dps nanocages can limit the degree of endotoxin-induced kidney injury. This was accomplished by amplifying the endogenous antioxidant property of Dps with addition of a dinuclear manganese cluster. Dps is the first-in-class protein cage nanoparticle that can be targeted to renal proximal tubules through glomerular filtration. In addition to its therapeutic potential, chemical and genetic engineering of Dps will offer a nanoplatform to advance our understanding of the physiology and pathophysiology of glomerular filtration and tubular endocytosis