Minicircle DNA Research Articles

Visualizing Spontaneous DNA Dynamics and its Role in Mismatch Recognition by Damage Recognition Protein Rad4

How DNA-binding proteins search for and identify their target sites buried amid a vast excess of non-target sites remains a central question in biology. This study is aimed at elucidating the mechanism by which DNA damage recognition protein XPC recognizes diverse DNA lesions, and initiates DNA repair. Structural studies of Rad4 (yeast ortholog of XPC) bound to model lesions showed that DNA is unwound and bent at the lesion site and the damaged nucleotides are flipped out. Previous studies using laser T-jump perturbation approaches indicated that Rad4 interrogates potential sites by unwinding DNA on ~100-500 μs and eventually flips out damaged nucleotides on ~5-10 ms. However, these ‘ensemble’ measurements could not capture intrinsic DNA deformability that is potentially critical in the ability of Rad4/XPC to distinguish damaged from undamaged sites. Previous studies were also limited to measurements on short, linear DNA oligomers, while DNA in our cells is typically bent and supercoiled, which is expected to have a profound impact on DNA damage recognition. In this study, I employed an innovative fluorescence lifetime approach with fluorescent probes exquisitely sensitive to DNA twisting/bending fluctuations and mapped the range of conformations accessible to mismatched DNA substrates of varying Rad4-binding specificities. My results reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity mismatched DNA, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound. Conversely, nonspecific mismatched DNA retained a largely homogeneous, B-DNA-like conformation. Surprisingly, even mismatched-DNA specifically bound to Rad4 remained highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. Using T-jump, I unveiled the rates of intrinsic DNA conformational dynamics in mismatched DNA. Remarkably, these rates remained unaltered in the presence of Rad4. Finally, I examined the effect of DNA bending strain on the intrinsic deformability at the mismatched sites by extending the fluorescence lifetime studies in the context of DNA minicircles. These studies revealed amplified distortions for both matched and mismatched sites in DNA minicircles, with further distortions detected upon Rad4 binding to mismatched sites. Taken together, my results strongly suggest that intrinsic DNA deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites.

Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors.

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4–8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.

Sensitivity and specificity of an in-clinic point-of-care PCR test for the diagnosis of canine leishmaniasis

Canine leishmaniasis is an important infectious disease worldwide. Although commonly used, antibody tests are often falsely negative, and in such cases direct detection of the pathogen, such as PCR, is necessary. However, PCR is only performed in specialized laboratories and not available in all localities. The aim of this study was to compare the sensitivity and specificity of an in-clinic point-of-care (ICPOC) PCR for the diagnosis of canine Leishmania spp. infection to those of a well characterized reference real-time PCR. In this study, 515 samples from 251 dogs (201 EDTA blood samples, 244 conjunctival swabs, 19 lymph node aspirates, and 51 bone marrow aspirates) were collected prospectively and analysed for the presence of Leishmania DNA using an ICPOC test. The results were compared to those of a reference real-time PCR for identification of Leishmania kinetoplast minicircle DNA. Sensitivity and specificity with 95% confidence interval (CI 95%) were determined.Specificity was 100% for all samples examined. Sensitivity was 57.1% (CI 95%, 34.0–78.2) in bone marrow aspirates, 58.8% (CI 95%, 32.9–81.6) in lymph node aspirates, 46.9% (CI 95%, 32.5–61.7) in conjunctival swabs, and 10.0% (CI 95%, 3.3–21.8) in blood. The ICPOC PCR was easy to perform and was reliable in the case of positive test results. A negative result, however, did not exclude infection and therefore requires further diagnostics.

The Plastid Genome in Cladophorales Green Algae Is Encoded by Hairpin Chromosomes

Virtually all plastid (chloroplast) genomes are circular double-stranded DNA molecules, typically between 100 and 200 kb in size and encoding circa 80-250 genes. Exceptions to this universal plastid genome architecture are very few and include the dinoflagellates, where genes are located on DNA minicircles. Here we report on the highly deviant chloroplast genome of Cladophorales green algae, which is entirely fragmented into hairpin chromosomes. Short- and long-read high-throughput sequencing of DNA and RNA demonstrated that the chloroplast genes of Boodlea composita are encoded on 1- to 7-kb DNA contigs with an exceptionally high GC content, each containing a long inverted repeat with one or two protein-coding genes and conserved non-coding regions putatively involved in replication and/or expression. We propose that these contigs correspond to linear single-stranded DNA molecules that fold onto themselves to form hairpin chromosomes. The Boodlea chloroplast genes are highly divergent from their corresponding orthologs, and display an alternative genetic code. The origin of this highly deviant chloroplast genome most likely occurred before the emergence of the Cladophorales, and coincided with an elevated transfer of chloroplast genes tothe nucleus. A chloroplast genome that is composed only of linear DNA molecules is unprecedented among eukaryotes, and highlights unexpected variation in plastid genome architecture.

Trypanosoma cruzi strain TcIV infects raccoons from Illinois.

BACKGROUNDThe northern limits of Trypanosoma cruzi across the territory of the United States remain unknown. The known vectors Triatoma sanguisuga and T. lecticularia find their northernmost limits in Illinois; yet, earlier screenings of those insects did not reveal the presence of the pathogen, which has not been reported in vectors or reservoir hosts in this state.OBJECTIVESFive species of medium-sized mammals were screened for the presence of T. cruzi.METHODSGenomic DNA was isolated from heart, spleen and skeletal muscle of bobcats (Lynx rufus, n = 60), raccoons (Procyon lotor, n = 37), nine-banded armadillos (Dasypus novemcinctus, n = 5), Virginia opossums (Didelphis virginiana, n = 3), and a red fox (Vulpes vulpes). Infections were detected targeting DNA from the kinetoplast DNA minicircle (kDNA) and satellite DNA (satDNA). The discrete typing unit (DTU) was determined by amplifying two gene regions: the Spliced Leader Intergenic Region (SL), via a multiplex polymerase chain reaction, and the 24Sα ribosomal DNA via a heminested reaction. Resulting sequences were used to calculate their genetic distance against reference DTUs.FINDINGS18.9% of raccoons were positive for strain TcIV; the rest of mammals tested negative.MAIN CONCLUSIONSThese results confirm for the first time the presence of T. cruzi in wildlife from Illinois, suggesting that a sylvatic life cycle is likely to occur in the region. The analyses of sequences of SL suggest that amplicons resulting from a commonly used multiplex reaction may yield non-homologous fragments.

Targeted Delivery of CRISPR/Cas9-Mediated Cancer Gene Therapy via Liposome-Templated Hydrogel Nanoparticles.

Due to its simplicity, versatility, and high efficiency, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has emerged as one of the most promising approaches for treatment of a variety of genetic diseases, including human cancers. However, further translation of CRISPR/Cas9 for cancer gene therapy requires development of safe approaches for efficient, highly specific delivery of both Cas9 and single guide RNA to tumors. Here, novel core-shell nanostructure, liposome-templated hydrogel nanoparticles (LHNPs) that are optimized for efficient codelivery of Cas9 protein and nucleic acids is reported. It is demonstrated that, when coupled with the minicircle DNA technology, LHNPs deliver CRISPR/Cas9 with efficiency greater than commercial agent Lipofectamine 2000 in cell culture and can be engineered for targeted inhibition of genes in tumors, including tumors the brain. When CRISPR/Cas9 targeting a model therapeutic gene, polo-like kinase 1 (PLK1), is delivered, LHNPs effectively inhibit tumor growth and improve tumor-bearing mouse survival. The results suggest LHNPs as versatile CRISPR/Cas9-delivery tool that can be adapted for experimentally studying the biology of cancer as well as for clinically translating cancer gene therapy.

Minicircle Versus Plasmid DNA Delivery by Receptor-Targeted Polyplexes.

Due to its minimal size and lack of bacterial backbone sequences, minicircle (MC) DNA presents a promising alternative to plasmid DNA (pDNA) for non-viral gene delivery in terms of biosafety and improved gene transfer. Here, luciferase pDNA (pCMV-luc) and analogous MC DNA (MC07.CMV-luc) were formulated into polyplexes with c-Met targeted, PEG-shielded sequence-defined oligoaminoamides, or linear PEI (linPEI) as standard transfection agent. Distinct physicochemical and biological characteristics were observed for polyplexes formed with either pDNA or MC DNA as vectors. The carriers were found to dominate the shape of polyplexes, whereas the DNA type was decisive for the nanoparticle size. c-Met-targeted, tyrosine trimer-containing polyplexes were optimized into compacted rod structures with a size of 65-100 nm for pDNA and 35-40 nm for MC. Notably, these MC polyplexes display a lack of cell cycle dependence of transfection and a ∼200-fold enhanced gene transfer efficiency in c-Met-positive DU145 prostate carcinoma cultures over their tyrosine-free pDNA analogues.

Novel PCR primers to diagnose visceral leishmaniasis using peripheral blood, spleen or bone marrow aspirates.

To establish a suitable method of diagnosis of visceral leishmaniasis (VL) using peripheral blood, spleen or bone marrow aspirates. Peripheral blood, bone marrow and spleen aspirate samples were collected from clinically suspected VL patients (n=26). A new PCR primer pair (MK1F/R) was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani, and Leishmania infantum, and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions. Test was validated by comparing with several other diagnostic methods. The designed primer set showed 100% specificity and 98% sensitivity in detecting VL using blood samples, when compared with more invasive samples: bone marrow or spleen aspirates. The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample, peripheral blood instead of bone marrow or spleen aspirates.

Generation of skeletal myogenic progenitors from human pluripotent stem cells using non-viral delivery of minicircle DNA

Currently, the most efficient and promising approach for generating large numbers of engraftable human skeletal myogenic progenitors from pluripotent stem cells requires the conditional in vitro overexpression of PAX7 using lentiviral vectors. Because a non-integrating approach would be preferable to eliminate or minimize the risk associated with random genomic integration, here we investigate whether transient expression of PAX7 using minicircle DNA would enable the generation of functional pluripotent stem cell-derived myogenic progenitors, equivalent to those generated by lentivirus. Our results demonstrate that upon multiple transfections, the minicircle approach allows for the scalable generation of myogenic progenitors and these undergo efficient terminal differentiation in vitro. However, transplantation of minicircle-generated myogenic progenitors resulted in limited engraftment. This is probably due to less efficient delivery and more transient PAX7 expression in these cultures since PAX7 downregulation is accompanied by high level of spontaneous differentiation. Thus, although the in vitro data shows that the minicircle approach could potentially replace the use of lentivirus, improvements in the transfection/expression system will be necessary before it will be a feasible strategy for the generation of myogenic progenitors for cell replacement therapy.

Murine melanoma cells incomplete reprogramming using non-viral vector.

The reprogramming of cancer cells into induced pluripotent stem cells or less aggressive cancer cells can provide a modern platform to study cancer-related genes and their interactions with cell environment before and after reprogramming. Herein, we aimed to investigate the reprogramming capacity of murine melanoma B16F10 cells. The B16F10 was transfected using non-viral circular DNA plasmid containing the genes Sox-2, Oct4, Nanog, Lin28 and green fluorescent protein (GFP). These cells were characterized by immunofluorescence, analysis RT-PCR and cell cycle. Our results demonstrated for the first time that reprogramming of B16F10 may be induced using non-viral minicircle DNA containing the four reprogramming factors Oct4, Sox2, Lin 28, Nanog (OSLN) and the GFP reporter gene. The resulting clones are composed by epithelioid cells. These cells display characteristics of cancer stem cells, thus expressing pluripotent stem cell markers and dividing asymmetrically and symmetrically. Reprogrammed B16F10 cells did not form teratomas; however, they showed the suppression of tumourigenic abilities characterized by a reduced tumour size, when compared with parental B16F10 cell line. In contrast to parental cell line that showed accumulation of the cells in S phase of cell cycle, the cells of reprogrammed clones are accumulated in G1 phase. Long-term cultivation of reprogrammed B16F10 cells induces regression of their reprogramming. Our data imply that in result of reprogramming of B16F10 cells less aggressive Murine Melanoma Reprogrammed Cancer Cells may be obtained. These cells represent an interesting model to study mechanism of cells malignancy as well as provide a novel tool for anti-cancer drugs screening.

Influence of DNA sequence on the structure of minicircles under torsional stress.

The sequence dependence of the conformational distribution of DNA under various levels of torsional stress is an important unsolved problem. Combining theory and coarse-grained simulations shows that the DNA sequence and a structural correlation due to topology constraints of a circle are the main factors that dictate the 3D structure of a 336 bp DNA minicircle under torsional stress. We found that DNA minicircle topoisomers can have multiple bend locations under high torsional stress and that the positions of these sharp bends are determined by the sequence, and by a positive mechanical correlation along the sequence. We showed that simulations and theory are able to provide sequence-specific information about individual DNA minicircles observed by cryo-electron tomography (cryo-ET). We provided a sequence-specific cryo-ET tomogram fitting of DNA minicircles, registering the sequence within the geometric features. Our results indicate that the conformational distribution of minicircles under torsional stress can be designed, which has important implications for using minicircle DNA for gene therapy.

Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles.

Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggests that DNA bending causes the modulation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we have developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been rotationally phased with respect to the histone octamer by TG motifs. We have used this system to show that the nucleosome dramatically modulates CPD formation in a T11-tract that covers one full turn of the nucleosome helix at seven different SHLs, and that the position of maximum CPD formation at all locations is shifted to the 5΄-side of that found in mixed-sequence nucleosomes. We also show that an 80-mer minicircle DNA using the same TG-motifs faithfully reproduces the CPD pattern in the nucleosome, indicating that it is a good model for protein-free rotationally phased bent DNA of the same curvature as in a nucleosome, and that bending is modulating CPD formation.

Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 during Helicase-Coupled DNA Synthesis by the Herpes Polymerase

The herpes helicase-primase (UL5-UL8-UL52) very inefficiently unwinds double-stranded DNA. To better understand the mechanistic consequences of this inefficiency, we investigated protein displacement activity by UL5-UL8-UL52, as well as the impact of coupling DNA synthesis by the herpes polymerase with helicase activity. While the helicase can displace proteins bound to the lagging strand template, bound proteins significantly impede helicase activity. Remarkably, UL5-UL8-UL52, an extremely inefficient helicase, disrupts the exceptionally tight interaction between streptavidin and biotin on the lagging strand template. It also unwinds DNA containing streptavidin bound to the leading strand template, although it does not displace the streptavidin. These data suggest that the helicase may largely or completely wrap around the lagging strand template, with minimal interactions with the leading strand template. We utilized synthetic DNA minicircles to study helicase activity coupled with the herpes polymerase-processivity factor (UL30-UL42). Coupling greatly enhances unwinding of DNA, although bound proteins still inhibit helicase activity. Surprisingly, while UL30-UL42 and two noncognate polymerases (Klenow Fragment and T4 DNA polymerase) all stimulate unwinding of DNA by the helicase, the isolated UL30 polymerase (i.e., no UL42 processivity factor) binds to the replication fork but in a manner that is incompetent in terms of coupled helicase-polymerase activity.

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

BackgroundLeishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area.MethodsDNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed.ResultsThe qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples.ConclusionsA new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.

Adipose-derived stem cell sheets functionalized by hybrid baculovirus for prolonged GDNF expression and improved nerve regeneration.

Peripheral nerve regeneration requires coordinated functions of supporting cells (e.g. Schwann cells) and neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF), but nerve regeneration is usually far from complete. Here we constructed a Cre/loxP-based hybrid baculovirus (BV) vector which enabled intracellular formation of episomal DNA minicircle for effective transduction of rat adipose-derived stem cells (ASCs) and prolonged expression of functional GDNF capable of recruiting Schwann cells. The GDNF expression persisted for >20 days with the peak level (≈128ng/ml) tremendously exceeding the picogram levels of GDNF secreted by neuroprogenitor cells. We further developed a facile method to fabricate and transduce cell sheets composed of undifferentiated ASCs in 2 days, without the need of thermo-responsive polymer commonly used for cell sheet fabrication. Implantation of the hybrid BV-engineered, GDNF-expressing ASCs sheets into sciatic nerve transection site in rats significantly improved the nerve repair, as judged from the enhanced functional recovery, nerve reinnervation, electrophysiological functionality, Schwann cells proliferation/infiltration, axon regeneration, myelination and angiogenesis. The hybrid BV is able to functionalize ASCs sheets by intracellular episomal DNA minicircle formation that circumvents undesired gene integration, and the ASCs sheets fabrication is rapid and simple. These data and features implicate the potentials of ASCs sheets functionalized by the hybrid BV for peripheral nerve regeneration.

Minicircle Kinetoplast Genome of Insect Trypanosomatid Leptomonas pyrrhocoris.

We present here the structure of a minicircle population based on transcriptome sequencing of Leptomonas pyrrhocoris. We show that minicircle DNA molecules are dimeric. As in dixenous species, the entire molecule of minicircle DNA is transcribed. This is the first minicircle transcriptome of monoxenous trypanosomatid species determined using NGS technology.

Domestic dogs as reservoir hosts for Leishmania donovani in the southernmost Western Ghats in India

The peripheral blood samples from domestic dogs (n=47) and wild rats (n=25) in the Kani Tribe settlements, located southernmost part of the Western Ghats, Thiruvananthapuram district, Kerala, India were examined for Leishmania infection. This area is known for cases of leishmaniasis with cutaneous manifestations and sandfly abundance. The tribes domesticate dogs to protect them from untoward activities of wild animals. Leishmania donovani parasite DNA was detected only from 6.4% (n=3) of the blood samples collected from the domestic dogs by amplification of the diagnostic kinetoplast mini-circle DNA and PCR-RFLP analysis of the UTR region of heat shock protein 70 (hsp70) gene. None of the blood samples collected from rats was positive. Through sequencing, L. donovani infection among dogs was confirmed. The DNA sequences generated for hsp70 were deposited with the GenBank. The GenBank accession numbers of these samples are KR905363, KR905364 and KR905365 for hsp70 genes. The results indicated that the DNA isolates from dog blood samples matched precisely with that of our earlier isolates from skin lesions of Kani tribes and also from P. argentipes vector. Thus, the role of dogs as reservoirs for L. donovani parasite in the Kani tribe settlements is confirmed.

Minicircle classes heterogeneity within the TcIII and TcIV discrete typing units of Trypanosoma cruzi

The taxon Trypanosoma cruzi, causative agent of Chagas disease, is composed of several discrete typing units (DTUs) named TcI-TcVI, and Tcbat. The history of the taxon T. cruzi is known, even though several controversial aspects remain as the relationships between TcIII and TcIV. We analyzed cloned T. cruzi stocks pertaining to the seven DTUs by filter hybridization tests of PCR amplicons from minicircle variable regions and kinetoplast DNA probes. Minicircle DNA blots from the cloned stocks and filter hybridization with one TcI, one TcII, one TcV, one TcVI, three TcIII, one TcIV from North America and one TcIV kinetoplast DNA probes from South America revealed minicircle variable region cross-reaction in some T. cruzi DTUs probed. TcIII was heterogeneous in minicircle class composition, even though two TcIII probes revealed that a small fraction of minicircles cross-hybridized with the minicircles from the TcIII, TcV and TcVI DTUs. The minicircles of TcIV from North America cross-reacted only with TcIV from North America but not with TcIV stocks from Brazil and Bolivia. The results on minicircle cross-hybridizations are discussed in the context of RNA editing, mitochondrial function in T. cruzi DTUs.

Abstract B49: Sustained IFNalpha treatment inhibits metastasis in 4T1 mammary carcinoma and correlates with decreasing myeloid-derived suppressor cells

Abstract In order to model pharmacokinetics and pharmacodynamics of pegylated IFNalpha, we utilized hydrodynamic delivery technology using minicircle DNA vectors encoding universal IFNalpha as a rapid and cost-effective method to obtain sustained levels of serum IFNalpha2alpha1. This approach is particularly useful for delivering secreted molecules, such as cytokines, as transfected hepatocytes secrete the transgene product in a paracrine manner providing a serum exposure profile resembling that reported in PEG-intron-treated patients. We found that sustained exposure of IFNalpha2alpha1 induced inhibition of metastasis. Primary 4T1 tumor growth was partially inhibited, while significant decreases in pulmonary metastatic burden were consistently observed. Further evidence that the observed inhibition of metastasis was independent of potential effects on the primary tumor was tested by resecting the primary tumor prior to treatment, which did not alter the outcome. Importantly, sustained IFNalpha2alpha1 treatment correlated with a significant increase in the survival of tumor-bearing animals. IFNalpha effects are plieotropic resulting in potential activation of numerous immune cell types associated with both innate and adaptive immunity. However, the mechanisms associated with IFNalpha-induced inhibition of lung metastasis were largely engendered by innate immune cells since treatment in BALB/c mice deficient for T and B cells did not affect the capacity of IFNalpha to inhibit pulmonary metastasis when compared to wild-type animals. The importance of NK cells has recently been reported as an important innate component for IFNalpha induced reduction of metastatic burden. However, little is known about IFNalpha-regulation of other innate cell types that may contribute to the inhibition of tumor metastasis. Here we report IFNalpha2alpha1 treatment induced marked decreases in 4T1-induced expansion and activation of immature CD11b+ Gr-1+ myeloid cells (aka: MDSC), which have been reported to contribute to metastatic potential. These results correlated with similar decreases in metastatic burden in mice following chlodronate-liposome depletion of CD11b+ Gr-1+ myeloid cells. Citation Format: Christina Kochel, Kalyan Pande, Roanna Ueda, Dan Gorman, Drake Laface. Sustained IFNalpha treatment inhibits metastasis in 4T1 mammary carcinoma and correlates with decreasing myeloid-derived suppressor cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B49.

Intraspecific genetic variability in a population of Moroccan Leishmania infantum revealed by PCR-RFLP of kDNA minicircles

In Morocco, Leishmania infantum is the main etiologic agent of human and canine visceral leishmaniasis (VL). This species has been proven to be an opportunistic agent in HIV+ patients and is also responsible of sporadic cutaneous leishmaniasis (CL).This work aims to evaluate the genetic variability of Moroccan L. infantum strains based on PCR-RFLP analysis of the kinetoplastid DNA (kDNA) minicircles. A total of 75 DNA samples extracted from positive Giemsa-stained smears (n=32) and from L. infantum cultures (n=43) was studied. The samples have been taken from VL patients infected (n=7) or not (n=56) by HIV, patients with CL (n=2) and finally from infected dogs (n=10). An hypervariable region of kDNA was amplified using the primers MC1 and MC2; the PCR products were digested separately by a panel of nine restriction enzymes. The presence or absence of restriction fragments was scored in a binary matrix and the SplitsTree4 software was used for the construction of a Neighbor-Net network. Moroccan L. infantum population showed an important level of variability with the identification of 6 genotypes. For each genotype a PCR product was sequenced, confirming the presence of all the expected restriction sites. The predominant profile was the genotype B. A new genotype, named Q was detected for the first time, whereas the four other genotypes (G, K, N and O) were reported sporadically in the Mediterranean basin. The Neighbor-Net network segregates our L. infantum population into 3 clusters: Cluster I includes genotype B, cluster II grouping the genotypes O, Q and G and finally the cluster III contains the genotype N. The kDNA-PCR-RFLP assay is suitable for use directly on biological samples; it reveals an important degree of genetic variability among L. infantum strains even those belonging to the same zymodeme what is of great epidemiological interest.