Publications by authors named "Qixin Leng"

43 Publications

Targeting KDM1B-dependent miR-215-AR-AGR2-axis promotes sensitivity to enzalutamide-resistant prostate cancer.

Cancer Gene Ther 2021 Apr 14. Epub 2021 Apr 14.

Research Center of Medical Sciences, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China.

Post-translational modifications of histones by histone demethylases plays an important role in the regulation of gene transcription and are implicated in cancers. Castrate resistant prostate cancer (CRPC) is often driven by constitutively active androgen receptor and commonly becomes resistant to established hormonal therapy strategies such as enzalutamide as a result. However, the role of KDM1B involved in next generation anti-enzalutamide resistance and the mechanisms of KDM1B regulation are poorly defined. Here, we show that KDM1B is upregulated and correlated with prostate cancer progression and poor prognosis. Downregulation of miR-215 is correlated with overexpression of KDM1B in enzalutamide-resistant prostate cancer cells, which promotes AR-dependent AGR2 transcription and regulates the sensitivity to next generation AR-targeted therapy. Inhibition of KDM1B significantly inhibits prostate tumor growth and improves enzalutamide treatments through AGR2 suppression. Our studies demonstrate inhibition of KDM1B can offer a viable therapeutic option to overcome enzalutamide resistance in tumors with deregulated miR-215-KDM1B-AR-AGR2 signaling axis.
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http://dx.doi.org/10.1038/s41417-021-00332-6DOI Listing
April 2021

Microbiota Biomarkers for Lung Cancer.

Diagnostics (Basel) 2021 Feb 27;11(3). Epub 2021 Feb 27.

Department of Pathology, School of Medicine, University of Maryland, 10 S. Pine St., Baltimore, MD 21201, USA.

Non-small cell lung cancer (NSCLC) is the number one cancer killer and its early detection can reduce mortality. Accumulating evidences suggest an etiopathogenic role of microorganisms in lung tumorigenesis. Certain bacteria are found to be associated with NSCLC. Herein we evaluated the potential use of microbiome as biomarkers for the early detection of NSCLC. We used droplet digital PCR to analyze 25 NSCLC-associated bacterial genera in 31 lung tumor and the paired noncancerous lung tissues and sputum of 17 NSCLC patients and ten cancer-free smokers. Of the bacterial genera, four had altered abundances in lung tumor tissues, while five were aberrantly abundant in sputum of NSCLC patients compared with their normal counterparts (all < 0.05). Acidovorax and Veillonella were further developed as a panel of sputum biomarkers that could diagnose lung squamous cell carcinoma (SCC) with 80% sensitivity and 89% specificity. The use of Capnocytophaga as a sputum biomarker identified lung adenocarcinoma (AC) with 72% sensitivity and 85% specificity. The use of Acidovorax as a sputum biomarker had 63% sensitivity and 96% specificity for distinguishing between SCC and AC, the two major types of NSCLC. The sputum biomarkers were further validated for the diagnostic values in a different cohort of 69 NSCLC cases and 79 cancer-free controls. Sputum microbiome might provide noninvasive biomarkers for the early detection and classification of NSCLC.
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http://dx.doi.org/10.3390/diagnostics11030407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997424PMC
February 2021

Sensitive Detection of KRAS Mutations by Clustered Regularly Interspaced Short Palindromic Repeats.

Diagnostics (Basel) 2021 Jan 15;11(1). Epub 2021 Jan 15.

Department of Pathology, University of Maryland School of Medicine, 10 S. Pine St., Baltimore, MD 21201, USA.

Kirsten rat sarcoma viral oncogene (KRAS) is the isoform most frequently mutated in human tumors. Testing for activating KRAS mutations has important implications for diagnosis and the personalized medicine of cancers. The current techniques for detecting KRAS mutations have moderate sensitivity. The emerging clustered regularly interspaced short palindromic repeats (CRISPR) system shows great promise in the detection of nucleic acids and is revolutionizing medical diagnostics. This study aimed to develop CRISPR-Cas12a as a sensitive test to detect KRAS mutations. Serially diluted DNA samples containing KRAS mutations are subjected to CRISPR-Cas12a and polymerase chain reaction (PCR). CRISPR-Cas12a and PCR can specifically detect 0.01% and 0.1% mutant KRAS DNA in the presence of wild-type KRAS DNA, respectively. Twenty pairs of lung tumor and noncancerous lung tissues are tested by CRISPR-Cas12a, PCR, and direct sequencing. CRISPR-Cas12a could identify the G12C mutation in five of 20 tumor tissues, while both PCR and direct sequencing discovered the KRAS mutation in three of the five tumor tissues. Furthermore, the results of CRISPR-Cas12a for testing the mutation could be directly and immediately visualized by a UV light illuminator. Altogether, CRISPR-Cas12a has a higher sensitivity for the detection of KRAS mutations compared with PCR and sequencing analysis, and thus has diagnostic and therapeutic implications. Nevertheless, the technique needs to be validated for its clinical significance in a large and prospective study.
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http://dx.doi.org/10.3390/diagnostics11010125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830957PMC
January 2021

Location of a single histidine within peptide carriers increases mRNA delivery.

J Gene Med 2021 02 21;23(2):e3295. Epub 2020 Dec 21.

Department of Pathology, University Maryland School of Medicine, University of Maryland, Baltimore, MD, USA.

Background: Previously, we determined that four-branched histidine-lysine (HK) peptides were effective carriers of plasmids and small interfering RNA. In the present study, we compared several branched HK carriers and, in particular, two closely-related H3K4b and H3K(+H)4b peptides for their ability as carriers of mRNA. The H3K(+H)4b peptide differed from its parent analogue, H3K4b, by only a single histidine in each branch.

Methods: A series of four-branched HK peptides with varied sequences was synthesized on a solid-phase peptide synthesizer. The ability of these peptides to carry mRNA expressing luciferase to MDA-MB-231 cells was investigated. With gel retardation and heparin displacement assays, the stability of HK polyplexes was examined. We determined the intracellular uptake of HK polyplexes by flow cytometry and fluorescence microscopy. The size and polydispersity index of the polyplexes in several media were measured by dynamic light scattering.

Results: MDA-MB-231 cells transfected by H3K(+H)4b-mRNA polyplexes expressed 10-fold greater levels of luciferase than H3K4b polyplexes. With gel retardation and heparin displacement assays, the H3K(+H)4b polyplexes showed greater stability than H3K4b. Intracellular uptake and co-localization of H3K(+H)4b polyplexes within acidic endosomes were also significantly increased compared to H3K4b. Similar to H3K(+H)4b, several HK analogues with an additional histidine in the second domain of their branches were effective carriers of mRNA. When combined with DOTAP liposomes, H3K(+H)4b was synergistic in delivery of mRNA.

Conclusions: H3K(+H)4b was a more effective carrier of mRNA than H3K4b. Mechanistic studies suggest that H3K(+H)4b polyplexes were more stable than H3K4b polyplexes. Lipopolyplexes formed with H3K(+H)4b markedly increased mRNA transfection.
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http://dx.doi.org/10.1002/jgm.3295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900953PMC
February 2021

Identification of a novel differentially methylated region adjacent to in lung cancer cells using methyl-CpG binding domain protein-enriched genome sequencing.

Genome 2021 May 28;64(5):533-546. Epub 2020 Oct 28.

Department of Animal & Avian Sciences, University of Maryland, College Park, MD 20742, USA.

Lung cancer is the most common cancer worldwide. Epigenetic modifications like DNA methylation play fundamental roles in the dynamic process of lung cancer. The objective of this study was to use methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) to identify novel and high-confidence DNA methylation in lung tumor. We first compared the whole-genome DNA methylation of three lung cancer cell lines, including A549, H1299, and SK-MES-1, against BEAS-2B, a lung/bronchial normal epithelial cell line. We then used pyrosequencing and OneStep qMethyl kit methods to verify the results in the cell line specimens. MBD-Seq identified 279, 8046, and 22 887 differentially methylated regions (DMRs), respectively, with 120 common DMRs among three comparison groups. Three DMRs were consistent with the MBD-Seq results by both pyrosequencing and OneStep qMethyl validations. Furthermore, OneStep qMethyl kit was also performed for functional validation of these three potential DMRs in sputum DNA from clinical participants. We successfully identified one new DMR adjacent to . The novel DMR might have an important function in lung carcinogenesis. Further validation of the finding in clinical specimens of lung cancer patients and functional analysis of this novel DMR in the development of lung cancer through transcriptional silencing of are warranted.
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http://dx.doi.org/10.1139/gen-2020-0071DOI Listing
May 2021

A CRISPR Test for Rapidly and Sensitively Detecting Circulating EGFR Mutations.

Diagnostics (Basel) 2020 Feb 19;10(2). Epub 2020 Feb 19.

Department of Pathology, University of Maryland School of Medicine, 10 S. Pine St. Baltimore, MD 21201, USA.

The detection of EGFR mutations in circulating cell-free DNA can enable personalized therapy for cancer. The current techniques for detecting circulating EGFR mutations are expensive and time-consuming with moderate sensitivity. Emerging CRISPR is revolutionizing medical diagnostics and showing a great promise for nucleic acid detection. This study aims to develop CRISPR-Cas12a as a simple test to sensitively detect circulating EGFR mutations in plasma. Serially diluted samples of DNA containing heterozygous EGFR mutations (L858R and T790M) in wild-type genomic DNA are concurrently tested for the mutations by a CRISPR-Cas12a system and droplet digital PCR (ddPCR). The CRISPR-Cas12a system can detect both L858R and T790M with a limit of detection of 0.005% in less than three hours. ddPCR detects the mutations with a limit of detection of 0.05% for more than five hours. Plasma samples of 28 lung cancer patients and 20 cancer-free individuals are tested for the EGFR mutations by CRISPR-Cas12a system and ddPCR. The CRISPR-Cas12a system could detect L858R in plasma of two lung cancer patients whose tissue biopsies are positive for L858R, and one plasma sample of three lung cancer patients whose tissue biopsies are positive for T790M. ddPCR detects L858R in the same two plasm samples, however, does not detect T790M in any of the plasma samples. This proof of principle study demonstrates that the CRISPR-Cas12a system could rapidly and sensitively detect circulating EGFR mutations, and thus, has potential prognostic or therapeutic implications.
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http://dx.doi.org/10.3390/diagnostics10020114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168902PMC
February 2020

MicroRNA-based biomarkers for diagnosis of non-small cell lung cancer (NSCLC).

Thorac Cancer 2020 03 28;11(3):762-768. Epub 2020 Jan 28.

Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Background: The development of biomarkers for the early detection of non-small cell lung cancer (NSCLC) is clinically important. We have developed miRNA biomarkers in sputum and plasma, respectively, for NSCLC. Herein, we evaluate whether integrated analysis of the miRNAs across the different types of specimens could improve the early detection of NSCLC.

Methods: Using reverse transcription PCR, we determined expressions of two miRNAs (miRs-31-5p and 210-3p) in sputum and three miRNAs (miRs-21-5p, 210-3p, and 486-5p) in plasma of a training cohort of 76 NSCLC patients and 72 cancer-free smokers. The results were validated in a testing cohort of 56 NSCLC patients and 55 cancer-free smokers.

Results: The panels of two sputum miRNAs and three plasma miRNAs had 65.8-75.0% sensitivities and 83.3-87.5% specificities for diagnosis of NSCLC in the training cohort. The individual sputum or plasma miRNA panel had a higher sensitivity for squamous cell carcinoma or adenocarcinoma of the lung, respectively. From the miRNAs, we optimized an integrated panel of biomarkers consisting of two sputum miRNAs (miRs-31-5p and 210-3p) and one plasma miRNA (miR-21-5p) that had higher sensitivity (85.5%) and specificity (91.7%) for diagnosis of NSCLC compared with the individual panels alone. Furthermore, the performance of the integrated panel of biomarkers was independent of histology and stage of NSCLC, and patients' age, sex, and ethnicity. The performance of the integrated panel of biomarkers was confirmed in the testing cohort.

Conclusions: Integrating biomarkers across different body fluids would synergistically improve the early detection of NSCLC.

Key Points: Lung cancer is a heterogeneous disease and develops from complex aberrations. Integrating sputum and plasma miRNAs has higher accuracy than when they are used alone.
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http://dx.doi.org/10.1111/1759-7714.13337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049510PMC
March 2020

A CRISPR Test for Detection of Circulating Nuclei Acids.

Transl Oncol 2019 Dec 18;12(12):1566-1573. Epub 2019 Oct 18.

Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD, USA. Electronic address:

Emerging CRISPR-based nucleic acid detection shows great promise in molecular diagnosis of diseases. CRISPR-Cas12a can sensitively and specifically detect human papillomavirus (HPV) DNA in anal swabs. However, the current CRISPR-Cas12a system needs auxiliary and expensive equipment, which limit its application as a point-of-care (POC) diagnostic tool. This study aimed to develop CRISPR-Cas12a as a POC test to directly target plasma for circulating HPV DNA detection by immediately reading results with naked eyes. Cell-cultured supernatants of either HPV16- or 18-positive cancer cells were treated with lysis buffer followed by isothermal amplification without DNA isolation. Cas12a, crRNA, and fluorescent-biotin reporters were incubated with the lysates. Our data showed that integrating CRISPR-Cas12a with lateral-flow strips could directly and specifically detect HPV16 and 18 in the liquid samples with the same limit of detection (0.24 fM) as did polymerase chain reaction but requiring less time. Furthermore, the CRISPR-Cas12a system could rapidly detect presence of HPV16 and HPV18 in plasma samples of 13 of 14 and 3 of 10 the patients with histopathological diagnosis of cervical cancer, respectively. Therefore, a CRISPR-Cas12a-based POC system was developed for conveniently detecting circulating nuclei acid targets in body fluids without requiring technical expertise and ancillary machineries.
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http://dx.doi.org/10.1016/j.tranon.2019.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6807067PMC
December 2019

Targeting Cancer with Peptide RNAi Nanoplexes.

Methods Mol Biol 2019 ;1974:161-180

AparnaBio, Inc., Gaithersburg, MD, USA.

With the recent explosion of genomic information on the root causes of disease, there is an increased interest in nucleic acid therapeutics, including siRNA and gene therapy, all of which require delivery of highly charged nucleic acids from siRNA with a molecular weight of about 1.4 × 10 to plasmids with an approximate molecular weight of 2.0-3.0 × 10. This chapter describes the delivery of shRNA via plasmid or siRNA with a peptide-based carrier. We focus on the histidine-lysine peptide which serves as an example for other peptides and polymeric carrier systems. When the HK peptide and nucleic acids are mixed together and interact with one another through ionic and nonionic interactions, nanoplexes are formed. These nanoplexes, carrying either shRNA or siRNA that target oncogenes, provide promising options for the treatment of cancer. We describe methods of preparation and characterization of these nanoplexes using dynamic light scattering, zeta potential, and gel retardation assays. We also provide protocols for transfection in vitro and in vivo for these nanoplexes.
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http://dx.doi.org/10.1007/978-1-4939-9220-1_12DOI Listing
November 2019

Enhanced tumor uptake and activity of nanoplex-loaded doxorubicin.

Biochem Biophys Res Commun 2019 05 4;513(1):242-247. Epub 2019 Apr 4.

Department of Pathology, University Maryland School of Medicine, Baltimore, MD, 21201, United States. Electronic address:

Doxorubicin (Dox) has widespread use as a cancer chemotherapeutic agent, but Dox is limited by several side effects including irreversible cardiomyopathy. Although liposomal Dox formulations, such as Doxil, mitigate side effects, they do not prolong survival in many patients. As a result, efforts have continued to discover improved formulations of Dox. We previously found that a peptide-based nanoplex delivered plasmid DNA efficiently to tumors in murine models. Unlike the majority of nanoparticles that depend solely on enhanced permeability and retention (EPR) for their transport into the tumor, our peptide-based nanoplex has a potential advantage in that its uptake primarily depends on neuropilin-1 receptor targeting. Because Dox binds to DNA, we tested whether this delivery platform could effectively deliver Dox to tumors and reduce their size. The nanoplexes increased the levels of Dox in tumors by about 5.5-fold compared to aqueous (free) Dox controls. Consistent with enhanced levels in the tumor, the nanoplex-Dox treatment had significantly greater anti-tumor activity. Whereas low dose free Dox did not reduce the size of tumors compared to untreated controls, the low dose nanoplex-Dox reduced the size of tumors by nearly 55% (p < 0.001). The high dose nanoplex-Dox also inhibited the size of tumor significantly more than the comparable high-dose free Dox (p < 0.001). Furthermore, apoptosis and proliferation markers (Ki67) of tumors observed in the different treatment groups correlated with their ability to inhibit tumor size. This study shows the efficacy of an NRP-1 targeted nanoplexes to deliver Dox to tumors in vivo and lays the groundwork for more complex and effective formulations.
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http://dx.doi.org/10.1016/j.bbrc.2019.03.190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531033PMC
May 2019

Fucosylation genes as circulating biomarkers for lung cancer.

J Cancer Res Clin Oncol 2018 Nov 12;144(11):2109-2115. Epub 2018 Aug 12.

Department of Pathology, The University of Maryland School of Medicine, 10 South Pine Street, MSTF 7th Floor, Baltimore, MD, 21201-1192, USA.

Purpose: Fucosyltransferases (FUTs) catalyze fucosylation, which plays a central role in biological processes. Aberrant fucosylation is associated with malignant transformation. Here we investigated whether transcriptional levels of genes coding the FUTs in plasma could provide cell-free circulating biomarkers for lung cancer.

Methods: mRNA expression of all 13 Futs (Fut1-11, Pofut1, and Pofut2) was evaluated by PCR assay in 48 lung tumor tissues and the 48 matched noncancerous lung tissues, and plasma of 64 lung cancer patients and 32 cancer-free individuals to develop plasma Fut biomarkers. The developed plasma Fut biomarkers were validated in an independent cohort of 40 lung cancer patients and 20 controls for their diagnostic performance.

Results: Four of the 13 Futs showed a different transcriptional level in 48 lung tumor tissues compared with the 48 matched nonconscious tissues (all < 0.05). Two (Fut8, and Pofut1) of the four Futs had a higher plasma level in 64 lung cancer patients compared with 32 control subjects, and consistent with that in lung tissue specimens. Combined analysis of the two Futs produced 81% sensitivity and 86% specificity for diagnosis of lung cancer, and was independent of stage and histology of lung tumors. The diagnostic performance of the two plasma biomarkers was successfully validated in the different cohort of 40 lung cancer patients and 20 control individuals.

Conclusion: The fucosylation genes may provide new circulating biomarkers for the early detection of lung cancer.
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http://dx.doi.org/10.1007/s00432-018-2735-0DOI Listing
November 2018

A Plasma Long Noncoding RNA Signature for Early Detection of Lung Cancer.

Transl Oncol 2018 Oct 8;11(5):1225-1231. Epub 2018 Aug 8.

Department of Pathology, University of Maryland School of Medicine, 10 S. Pine St. Baltimore, MD 21201, USA. Electronic address:

The early detection of lung cancer is a major clinical challenge. Long noncoding RNAs (lncRNAs) have important functions in tumorigenesis. Plasma lncRNAs directly released from primary tumors or the circulating cancer cells might provide cell-free cancer biomarkers. The objective of this study was to investigate whether the lncRNAs could be used as plasma biomarkers for early-stage lung cancer. By using droplet digital polymerase chain reaction, we determined the diagnostic performance of 26 lung cancer-associated lncRNAs in plasma of a development cohort of 63 lung cancer patients and 33 cancer-free individuals, and a validation cohort of 39 lung cancer patients and 28 controls. In the development cohort, 7 of the 26 lncRNAs were reliably measured in plasma. Two (SNHG1 and RMRP) displayed a considerably high plasma level in lung cancer patients vs. cancer-free controls (all P < .001). Combined use of the plasma lncRNAs as a biomarker signature produced 84.13% sensitivity and 87.88% specificity for diagnosis of lung cancer, independent of stage and histological type of lung tumor, and patients' age and sex (all P > .05). The diagnostic value of the plasma lncRNA signature for lung cancer early detection was confirmed in the validation cohort. The plasma lncRNA signature may provide a potential blood-based assay for diagnosing lung cancer at the early stage. Nevertheless, a prospective study is warranted to validate its clinical value.
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http://dx.doi.org/10.1016/j.tranon.2018.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6089091PMC
October 2018

An integromic signature for lung cancer early detection.

Oncotarget 2018 May 15;9(37):24684-24692. Epub 2018 May 15.

Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

We previously developed three microRNAs (miRs-21, 210, and 486-5p), two long noncoding RNAs (lncRNAs) (SNHG1 and RMRP), and two fucosyltransferase (FUT) genes (FUT8 and POFUT1) as potential plasma biomarkers for lung cancer. However, the diagnostic performance of the individual panels is not sufficient to be used in the clinics. Given the heterogeneity of lung tumors developed from multifactorial molecular aberrations, we determine whether integrating the different classes of molecular biomarkers can improve diagnosis of lung cancer. By using droplet digital PCR, we analyze expression of the seven genes in plasma of a development cohort of 64 lung cancer patients and 33 cancer-free individuals. The panels of three miRNAs (miRs-21, 210, and 486-5p), two lncRNAs (SNHG1 and RMRP), and two FUTs (FUT8 and POFUT1) have a sensitivity of 81-86% and a specificity of 84-87% for diagnosis of lung cancer. From the seven genes, an integromic plasma signature comprising miR-210, SNHG1, and FUT8 is developed that produces higher sensitivity (95.45%) and specificity (96.97%) compared with the individual biomarker panels (all p<0.05). The diagnostic value of the signature was confirmed in a validation cohort of 40 lung cancer patients and 29 controls, independent of stage and histological type of lung tumor, and patients' age, sex, and smoking status (all p>0.05). The integration of the different categories of biomarkers might improve diagnosis of lung cancer.
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http://dx.doi.org/10.18632/oncotarget.25227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973873PMC
May 2018

A Direct Plasma miRNA Assay for Early Detection and Histological Classification of Lung Cancer.

Transl Oncol 2018 Aug 31;11(4):883-889. Epub 2018 May 31.

Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Electronic address:

Cell-free microRNAs in plasma provide circulating biomarkers for lung cancer. Most techniques for analysis of miRNAs require a large plasma volume to purify a sufficient RNA yield followed by complicated downstream processing. Small differences in the multiple procedures often cause large analytical variations and poor diagnostic values of the plasma biomarkers. Here we investigate whether directly quantifying plasma miRNAs without RNA purification could diagnose lung cancer. FirePlex assay was directly applied to 20 μl plasma of 56 lung cancer patients and 28 cancer free controls for quantifying 11 lung tumor-associated miRNAs. FirePlex assay is easier, less expensive and time-consuming for quantification of plasma miRNAs compared with conventional reverse transcription PCR with an equivalent analytic performance. From the lung tumor-associated miRNAs, a prediction model based on two miRNAs (miRs-205-5p and -210-3p) was developed, producing 78.6% sensitivity and 89.3% specificity for identifying lung cancer. The diagnostic value was independent of stage of lung tumor, and patients' age and sex (all P > 0.05). Furthermore, based on the same two miRNAs, additional prediction models were developed with 75.0% sensitivity and 89.3% specificity for diagnosis of lung squamous cell carcinoma, and 82.2% sensitivity and 89.3% specificity for lung adenocarcinoma. The direct plasma assay can improve the efficacy of miRNA assessment in a small plasma volume by reducing multiple procedure-associated analytical variables. The developed plasma miRNA biomarkers might be useful for the early detection and histological classification of lung cancer.
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http://dx.doi.org/10.1016/j.tranon.2018.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041566PMC
August 2018

Integrating Circulating Immunological and Sputum Biomarkers for the Early Detection of Lung Cancer.

Biomark Cancer 2018 13;10:1179299X18759297. Epub 2018 Feb 13.

Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA.

We have demonstrated that assessments of microRNA (miRNA) expressions in circulating peripheral blood mononucleated cell (PBMC) and sputum specimens, respectively, may help diagnose lung cancer. To assess the individual and combined analysis of the miRNAs across the different body fluids for lung cancer early detection, we analyse a panel of 3 sputum miRNAs (miRs-21, 31, and 210) and a panel of 2 PBMC miRNAs (miRs-19b-3p and 29b-3p) in a discovery cohort of 68 patients with lung cancer and 66 cancer-free smokers. We find that integrating 2 sputum miRNAs (miRs-31 and 210) and 1 PBMC miRNA (miR-19b-3p) has higher sensitivity (86.8%) and specificity (92.4%) compared with the individual panels. The synergistic value of the integrated panel of 3 biomarkers is confirmed in a validation cohort, independent of stage and histological type of lung cancer, and patients' age, sex, and ethnicity. Integrating circulating immunological and sputum biomarkers could improve the early detection of lung cancer.
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http://dx.doi.org/10.1177/1179299X18759297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815414PMC
February 2018

A plasma miRNA signature for lung cancer early detection.

Oncotarget 2017 Dec 5;8(67):111902-111911. Epub 2017 Dec 5.

Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

The early detection of lung cancer continues to be a major clinical challenge. Using whole-transcriptome next-generation sequencing to analyze lung tumor and the matched noncancerous tissues, we previously identified 54 lung cancer-associated microRNAs (miRNAs). The objective of this study was to investigate whether the miRNAs could be used as plasma biomarkers for lung cancer. We determined expressions of the lung tumor-miRNAs in plasma of a development cohort of 180 subjects by using reverse transcription PCR to develop biomarkers. The development cohort included 92 lung cancer patients and 88 cancer-free smokers. We validated the biomarkers in a validation cohort of 64 individuals comprising 34 lung cancer patients and 30 cancer-free smokers. Of the 54 miRNAs, 30 displayed a significant different expression level in plasma of the lung cancer patients cancer-free controls (all P < 0.05). A plasma miRNA signature (miRs-126, 145, 210, and 205-5p) with the best prediction was developed, producing 91.5% sensitivity and 96.2% specificity for lung cancer detection. Diagnostic performance of the plasma miRNA signature had no association with stage and histological type of lung tumor, and patients' age, sex, and ethnicity (all p > 0.05). The plasma miRNA signature was reproducibly confirmed in the validation cohort. The plasma miRNA signature may provide a blood-based assay for diagnosing lung cancer at the early stage, and thereby reduce the associated mortality and cost.
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http://dx.doi.org/10.18632/oncotarget.22950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762367PMC
December 2017

Targeted Delivery of siRNA Therapeutics to Malignant Tumors.

J Drug Deliv 2017 9;2017:6971297. Epub 2017 Nov 9.

Department of Pathology, University of Maryland School of Medicine, 10 S. Pine St., Baltimore, MD 21201, USA.

Over the past 20 years, a diverse group of ligands targeting surface biomarkers or receptors has been identified with several investigated to target siRNA to tumors. Many approaches to developing tumor-homing peptides, RNA and DNA aptamers, and single-chain variable fragment antibodies by using phage display, evolution, and recombinant antibody methods could not have been imagined by researchers in the 1980s. Despite these many scientific advances, there is no reason to expect that the ligand field will not continue to evolve. From development of ligands based on novel or existing biomarkers to linking ligands to drugs and gene and antisense delivery systems, several fields have coalesced to facilitate ligand-directed siRNA therapeutics. In this review, we discuss the major categories of ligand-targeted siRNA therapeutics for tumors, as well as the different strategies to identify new ligands.
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http://dx.doi.org/10.1155/2017/6971297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700508PMC
November 2017

A prediction model for distinguishing lung squamous cell carcinoma from adenocarcinoma.

Oncotarget 2017 Aug 11;8(31):50704-50714. Epub 2017 Apr 11.

Department of Pathology, the University of Maryland School of Medicine, Baltimore, Maryland, USA.

Accurate classification of squamous cell carcinoma (SCC) from adenocarcinoma (AC) of non-small cell lung cancer (NSCLC) can lead to personalized treatments of lung cancer. We aimed to develop a miRNA-based prediction model for differentiating SCC from AC in surgical resected tissues and bronchoalveolar lavage (BAL) samples. Expression levels of seven histological subtype-associated miRNAs were determined in 128 snap-frozen surgical lung tumor specimens by using reverse transcription-polymerase chain reaction (RT-PCR) to develop an optimal panel of miRNAs for acutely distinguishing SCC from AC. The biomarkers were validated in an independent cohort of 112 FFPE lung tumor tissues, and a cohort of 127 BAL specimens by using droplet digital PCR for differentiating SCC from AC. A prediction model with two miRNAs (miRs-205-5p and 944) was developed that had 0.988 area under the curve (AUC) with 96.55% sensitivity and 96.43% specificity for differentiating SCC from AC in frozen tissues, and 0.997 AUC with 96.43% sensitivity and 96.43% specificity in FFPE specimens. The diagnostic performance of the prediction model was reproducibly validated in BAL specimens for distinguishing SCC from AC with a higher accuracy compared with cytology (95.69 vs. 68.10%; < 0.05). The prediction model might have a clinical value for accurately discriminating SCC from AC in both surgical lung tumor tissues and liquid cytological specimens.
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http://dx.doi.org/10.18632/oncotarget.17038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584193PMC
August 2017

NRP1 transport of cancer therapeutics mediated by tumor-penetrating peptides.

Drugs Future 2017 Feb;42(2):95-104

Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

Whereas uptake of low molecular weight agents is generally inhibited in tumors due to high interstitial pressure, tumor uptake of macromolecules is increased due to enhanced permeability and retention (EPR). Small molecule drugs alone or incorporated in nanoparticles (NP) have largely been dependent on such physical tumor uptake (passive) for therapeutic activity. Although passive targeted NP such as Stealth Liposomal Doxorubicin (Doxil ®) are effective with improved safety, drug delivery to tumors is still significantly limited. To improve tumor delivery and efficacy, tumor-penetrating peptides (TPP), which contain sequences that target the tumor and activate the neuropilin-1 receptor (NRP1), have either been co-administered with or conjugated to both small and large therapeutic molecules. In this review, we will discuss TPP-mediated therapeutics which target the NRP1 transport system of tumors.
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http://dx.doi.org/10.1358/dof.2017.042.02.2564106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461880PMC
February 2017

A classifier integrating plasma biomarkers and radiological characteristics for distinguishing malignant from benign pulmonary nodules.

Int J Cancer 2017 09 21;141(6):1240-1248. Epub 2017 Jun 21.

Department of Pathology, University of Maryland School of Medicine, Baltimore, MD.

Lung cancer is primarily caused by cigarette smoking and the leading cancer killer in the USA and across the world. Early detection of lung cancer by low-dose CT (LDCT) can reduce the mortality. However, LDCT dramatically increases the number of indeterminate pulmonary nodules (PNs), leading to overdiagnosis. Having a definitive preoperative diagnosis of malignant PNs is clinically important. Using microarray and droplet digital PCR to directly profile plasma miRNA expressions of 135 patients with PNs, we identified 11 plasma miRNAs that displayed a significant difference between patients with malignant versus benign PNs. Using multivariate logistic regression analysis of the molecular results and clinical/radiological characteristics, we developed an integrated classifier comprising two miRNA biomarkers and one radiological characteristic for distinguishing malignant from benign PNs. The classifier had 89.9% sensitivity and 90.9% specificity, being significantly higher compared with the biomarkers or clinical/radiological characteristics alone (all p < 0.05). The classifier was validated in two independent sets of patients. We have for the first time shown that the integration of plasma biomarkers and radiological characteristics could more accurately identify lung cancer among indeterminate PNs. Future use of the classifier could spare individuals with benign growths from the harmful diagnostic procedures, while allowing effective treatments to be immediately initiated for lung cancer, thereby reduces the mortality and cost. Nevertheless, further prospective validation of this classifier is warranted.
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http://dx.doi.org/10.1002/ijc.30822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526452PMC
September 2017

Silver adducts of four-branched histidine rich peptides exhibit synergistic antifungal activity.

Biochem Biophys Res Commun 2016 09 4;477(4):957-962. Epub 2016 Jul 4.

Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Electronic address:

Previously, a four branched histidine-lysine rich peptide, H3K4b, was shown to demonstrate selective antifungal activity with minimal antibacterial activity. Due to the potential breakdown from proteases, H3K4b was further evaluated in the current study by varying the D- and l-amino acid content in its branches. Whereas analogues of H3K4b that selectively replaced l-amino acids (H3k4b, h3K4b) had improved antifungal activity, the all d-amino acid analogue, h3k4b, had reduced activity, suggesting that partial breakdown of the peptide may be necessary. Moreover, because histidines form coordination bonds with the silver ion, we examined whether silver adducts can be formed with these branched histidine-lysine peptides, which may improve antifungal activity. For Candida albicans, the silver adduct of h3K4b or H3k4b reduced the MIC compared to peptide and silver ions alone by 4- and 5-fold, respectively. For Aspergillus fumigatus, the silver adducts showed even greater enhancement of activity. Although the silver adducts of H3k4b or h3K4b showed synergistic activity, the silver adduct with the all l-amino acid H3K4b surprisingly showed the greatest synergistic and growth inhibition of A. fumigatus: the silver adduct of H3K4b reduced the MIC compared to the peptide and silver ions alone by 30- and 26-fold, respectively. Consistent with these antifungal efficacy results, marked increases in free oxygen radicals were produced with the H3K4b and silver combination. These studies suggest that there is a balance between stability and breakdown for optimal antifungal activity of the peptide alone and for the peptide-silver adduct.
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http://dx.doi.org/10.1016/j.bbrc.2016.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968200PMC
September 2016

The neuropilin-1 receptor mediates enhanced tumor delivery of H2K polyplexes.

J Gene Med 2016 Jul;18(7):134-44

Department of Pathology, University Maryland School of Medicine, Baltimore, MD, USA.

Background: Promising plasmid-based treatments have limited value without an effective delivery system. Recently, the linear H2K with a repeating -KHHK- pattern was determined to be an effective plasmid carrier to tumor xenografts in vivo. Although unpacking of the H2K polyplex within the tumor may have a role, the mechanism for the enhanced efficacy remains unclear.

Methods: After solid-phase synthesis of linear and branched histidine-lysine (HK) peptide carriers of plasmids, the peptides were compared for their ability to lyse endosomes with a red blood cell model and to transfect MDA-MB-435 xenografts in the presence or absence of neuropilin-1 receptor (NRP-1) antibodies. To examine stability, polyplexes were incubated with trypsin or NaCl and then analyzed by electrophoresis.

Results: After screening peptides with a model for endosomal lysis at two pHs, the 33-mer H3K peptide lysed red blood cells effectively at the lower pH. Combining H3K and H2K peptides as carriers of plasmids expressing luciferase were more effective than H2K alone. Based on the repeating -KHHK- sequences of H2K, we studied whether the widespread gene expression in the tumor may be mediated by NRP-1. By blocking NRP-1 in tumor-bearing mice, luciferase activity in tumors delivered by HK polyplexes was reduced by 96%, whereas activity in normal tissues was minimally reduced.

Conclusions: Combining an endosomolytic peptide, H3K, with H2K polyplexes as a carrier further enhanced transfection in vivo. Moreover, the widespread distribution of H2K polyplexes is mediated by NRP-1, suggesting that transcytosis of these polyplexes through the tumor endothelium may lead to efficient transfection. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/jgm.2886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948292PMC
July 2016

Increased tumor distribution and expression of histidine-rich plasmid polyplexes.

J Gene Med 2014 Sep-Oct;16(9-10):317-28

Department of Pathology, University Maryland School of Medicine, Baltimore, MD, USA.

Background: Selecting nonviral carriers for in vivo gene delivery is often dependent on determining the optimal carriers from transfection assays in vitro. The rationale behind this in vitro strategy is to cast a net sufficiently wide to identify the few effective carriers of plasmids for in vivo studies. Nevertheless, many effective in vivo carriers may be overlooked by this strategy because of the marked differences between in vitro and in vivo assays.

Methods: After solid-phase synthesis of linear and branched histidine/lysine (HK) peptides, the two peptide carriers were compared for their ability to transfect MDA-MB-435 tumor cells in vitro and then in vivo.

Results: By contrast to their transfection activity in vitro, the linear H2K carrier of plasmids was far more effective in vivo compared to the branch H2K4b. Surprisingly, negatively-charged polyplexes formed by the linear H2K peptide gave higher transfection in vivo than did those with a positive surface charge. To examine the distribution of plasmid expression within the tumor from H2K polyplexes, we found widespread expression by immunohistochemical staining. With a fluorescent tdTomato expressing-plasmid, we confirmed a pervasive distribution and gene expression within the tumor mediated by the H2K polyplex.

Conclusions: Although mechanisms underlying the efficiency of gene expression are probably multifactorial, unpacking of the H2K polyplex within the tumor appears to have a significant role. Further development of these H2K polyplexes represents an attractive approach for plasmid-based therapies of cancer.
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http://dx.doi.org/10.1002/jgm.2807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4242722PMC
June 2015

Direct observation of dynamic mechanical regulation of DNA condensation by environmental stimuli.

Angew Chem Int Ed Engl 2014 Sep 21;53(40):10631-5. Epub 2014 Aug 21.

Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742 (USA).

Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single-molecule mechanical properties of DNA condensed using 19-mer poly-L-lysine (PLL) or branched histidine-lysine (HK) peptides. Force-extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.
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http://dx.doi.org/10.1002/anie.201403499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517097PMC
September 2014

Enhancement of antifungal activity by integrin-targeting of branched histidine rich peptides.

J Drug Target 2014 Jul 14;22(6):536-42. Epub 2014 Apr 14.

Aparna Biosciences Corp , Rockville, MD , USA .

The treatment of invasive candidiasis associated with growing numbers of immunocompromised patients remains a major challenge complicated by increasing drug resistance. A novel class of branched histidine-lysine (bHK) peptides has promising antifungal activity, and exhibits a mechanism similar to natural histatins, and thus may avoid drug resistance. The present studies evaluate ligand targeting of bHK peptides to fungal surface integrins by determining whether a cyclic RGD (cRGD) peptide with a large PEG linker could enhance bHK peptide antifungal activity. Whereas conjugates containing only the PEG linker reduced bHK peptide activity, conjugates with the cRGD-PEG ligand resulted in marked enhancement of activity against Candida albicans. This study provides the first demonstration of benefit from ligand targeting of antifungal agents to fungal surface receptors.
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http://dx.doi.org/10.3109/1061186X.2014.905948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072455PMC
July 2014

Enhanced silencing and stabilization of siRNA polyplexes by histidine-mediated hydrogen bonds.

Biomaterials 2014 Jan 22;35(2):846-55. Epub 2013 Oct 22.

Department of Pathology, University of Maryland Baltimore, MSTF Building, 10 South Pine Street, Baltimore, MD 21201, United States; Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, United States. Electronic address:

Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagine-lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively (15)N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of Nδ1-H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5-1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.
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http://dx.doi.org/10.1016/j.biomaterials.2013.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920840PMC
January 2014

Surface-modified HK:siRNA nanoplexes with enhanced pharmacokinetics and tumor growth inhibition.

Biomacromolecules 2013 Mar 14;14(3):752-60. Epub 2013 Feb 14.

Department of Pathology, University of Maryland Baltimore, MSTF Building, 10 South Pine Street, Baltimore, MD 21201, USA.

We characterized in this study the pharmacokinetics and antitumor efficacy of histidine-lysine (HK):siRNA nanoplexes modified with PEG and a cyclic RGD (cRGD) ligand targeting αvβ3 and αvβ5 integrins. With noninvasive imaging, systemically administered surface-modified HK:siRNA nanoplexes showed nearly 4-fold greater blood levels, 40% higher accumulation in tumor tissue, and 60% lower luciferase activity than unmodified HK:siRNA nanoplexes. We then determined whether the surface-modified HK:siRNA nanoplex carrier was more effective in reducing MDA-MB-435 tumor growth with an siRNA targeting Raf-1. Repeated systemic administration of the selected surface modified HK:siRNA nanoplexes targeting Raf-1 showed 35% greater inhibition of tumor growth than unmodified HK:siRNA nanoplexes and 60% greater inhibition of tumor growth than untreated mice. The improved blood pharmacokinetic results and tumor localization observed with the integrin-targeting surface modification of HK:siRNA nanoplexes correlated with greater tumor growth inhibition. This investigation reveals that through control of targeting ligand surface display in association with a steric PEG layer, modified HK: siRNA nanoplexes show promise to advance RNAi therapeutics in oncology and potentially other critical diseases.
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http://dx.doi.org/10.1021/bm3018356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3595641PMC
March 2013

Buffering capacity and size of siRNA polyplexes influence cytokine levels.

Mol Ther 2012 Dec 2;20(12):2282-90. Epub 2012 Oct 2.

Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

Induction of cytokines by small interfering RNA (siRNA) polyplexes has been a significant concern of researchers attempting to minimize the toxicity of this promising therapy. Although cationic carriers of siRNA are known to increase cytokine levels, few systematic studies have been done to determine what properties of the carrier are important to modulate cytokines. Because branched histidine-lysine (HK) peptides are effective carriers of siRNA and their sequence can be readily modified, we selected this class of carrier to determine which sequences of the peptide were important for cytokine induction. With the use of peripheral blood mononuclear cells (PBMCs), the HK peptide with a higher number of histidines (H3K(+H)4b) in complex with siRNA induced lower levels of cytokines compared with other HK (e.g., H2K4b, H3K4b, H3K(+N)4b) siRNA nanoplexes. Notably, these peptides' siRNA polyplexes showed a similar pattern of cytokine induction when injected intravenously in a mouse model, i.e., the HK with higher content of histidines induced cytokines the least. As indicated by the pH-sensitive dye within acidic endosomes, the greater pH-buffering capacity of H3K(+H)4b compared with other HK peptides may explain why cytokine levels were reduced. In addition to buffering capacity, the size of HK polyplexes markedly influenced cytokine production.
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http://dx.doi.org/10.1038/mt.2012.206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3519993PMC
December 2012

Zinc Finger Nucleases: Tailor-made for Gene Therapy.

Drugs Future 2012 Mar;37(3):183-196

Department of Pathology, University of Maryland School of Medicine, MSTF Building, 10 South Pine Street, Baltimore, MD 21201, USA ; Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742.

Genome editing with the use of zinc finger nucleases has been successfully applied to variety of a eukaryotic cells. Furthermore, the proof of concept for this approach has been extended to diverse animal models from to mice. Engineered zinc finger nucleases are able to target specifically and manipulate disease-causing genes through site-specific double strand DNA breaks followed by non-homologous end joining or homologous recombination mechanisms. Consequently, this technology has considerable flexibility that can result in either a gain or loss of function of the targeted gene. In addition to this flexibility, gene therapy by zinc finger nucleases may enable persistent long term gene modification without continuous transfection- a potential advantage over RNA interference or direct gene inhibitors. With systemic viral delivery systems, this gene-editing approach corrected the mutant factor IX in models of mouse hemophilia. Moreover, phase I clinical trials have been initiated with zinc finger nucleases in patients with glioblastoma and HIV. Thus, this emerging field has significant promise as a therapeutic strategy for human genetic diseases, infectious diseases and oncology. In this article, we will review recent advances and potential risks in zinc finger nuclease gene therapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3801298PMC
http://dx.doi.org/10.1358/dof.2012.037.03.1779022DOI Listing
March 2012

Vaccines targeting the neovasculature of tumors.

Vasc Cell 2011 Mar 8;3(1). Epub 2011 Mar 8.

Department of Pathology, University of Maryland Baltimore, MSTF Building, 10 South Pine Street, Baltimore, MD 21201, USA.

Angiogenesis has a critical role in physiologic and disease processes. For the growth of tumors, angiogenesis must occur to carry sufficient nutrients to the tumor. In addition to growth, development of new blood vessels is necessary for invasion and metastases of the tumor. A number of strategies have been developed to inhibit tumor angiogenesis and further understanding of the interplay between tumors and angiogenesis should allow new approaches and advances in angiogenic therapy. One such promising angiogenic approach is to target and inhibit angiogenesis with vaccines. This review will discuss recent advances and future prospects in vaccines targeting aberrant angiogenesis of tumors. The strategies utilized by investigators have included whole endothelial cell vaccines as well as vaccines with defined targets on endothelial cells and pericytes of the developing tumor endothelium. To date, several promising anti-angiogenic vaccine strategies have demonstrated marked inhibition of tumor growth in pre-clinical trials with some showing no observed interference with physiologic angiogenic processes such as wound healing and fertility.
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http://dx.doi.org/10.1186/2045-824X-3-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3061948PMC
March 2011
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