Kelly M Podetz-Pedersen

University of Minnesota Duluth, Duluth, Minnesota, United States

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Publications (14)54.56 Total impact

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    ABSTRACT: We previously utilized a Sleeping Beauty (SB) transposon mutagenesis screen to discover novel drivers of HCC. This approach identified recurrent mutations within the Dlk1-Dio3 imprinted domain, indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis. For the current study, we performed transcriptome and small RNA sequencing to profile gene expression in SB-induced HCCs in an attempt to clarify the genetic element(s) contributing to tumorigenesis. We identified strong induction of Retrotransposon-like 1 (Rtl1) expression as the only consistent alteration detected in all SB-induced tumors with Dlk1-Dio3 integrations, suggesting that Rtl1 activation serves as a driver of HCC. While previous studies have identified correlations between disrupted expression of multiple Dlk1-Dio3 domain members and HCC, we show here that direct modulation of a single domain member, Rtl1, can promote hepatocarcinogenesis in vivo. Overexpression of Rtl1 in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant (86%) tumor formation. Additionally, we detected overexpression of RTL1 in 30% of analyzed human HCC samples, indicating the potential relevance of this locus as a therapeutic target for patients. The Rtl1 locus is evolutionarily derived from the domestication of a retrotransposon. In addition to identifying Rtl1 as a novel driver of HCC, our study represents one of the first direct in vivo demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis.
    PLoS Genetics 04/2013; 9(4):e1003441. · 8.52 Impact Factor
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    ABSTRACT: Mucopolysaccharidosis type I (MPS I) is an autosomal recessive inherited disease caused by deficiency of the glycosidase α-L-iduronidase (IDUA). Deficiency of IDUA leads to lysosomal accumulation of glycosaminoglycans (GAG) heparan and dermatan sulfate and associated multi-systemic disease, the most severe form of which is known as Hurler syndrome. Since 1981, the treatment of Hurler patients has often included allogeneic BMT from a matched donor. However, mouse models of the disease were not developed until 1997. To further characterize the MPS-I mouse model and to study the effectiveness of BMT in these animals, we engrafted a cohort (n=33) of 4-8-week-old Idua(-/-) animals with high levels (88.4±10.3%) of wild-type donor marrow. Engrafted animals displayed an increased lifespan, preserved cardiac function, partially restored IDUA activity in peripheral organs and decreased GAG accumulation in both peripheral organs and in the brain. However, levels of GAG and GM3 ganglioside in the brain remained elevated in comparison to unaffected animals. As these results are similar to those observed in Hurler patients following BMT, this murine-transplantation model can be used to evaluate the effects of novel, more effective methods of delivering IDUA to the brain as an adjunct to BMT.
    Bone marrow transplantation 12/2011; 47(9):1235-40. · 3.00 Impact Factor
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    ABSTRACT: Artemis is an endonucleolytic enzyme involved in nonhomologous double-strand break repair and V(D)J recombination. Deficiency of Artemis results in a B- T- radiosensitive severe combined immunodeficiency, which may potentially be treatable by Artemis gene transfer into hematopoietic stem cells. However, we recently found that overexpression of Artemis after lentiviral transduction resulted in global DNA damage and increased apoptosis. These results imply the necessity of effecting natural levels of Artemis expression, so we isolated a 1 kilobase DNA sequence upstream of the human Artemis gene to recover and characterize the Artemis promoter (APro). The sequence includes numerous potential transcription factor-binding sites, and several transcriptional start sites were mapped by 5' rapid amplification of cDNA ends. APro and deletion constructs conferred significant reporter gene expression in vitro that was markedly reduced in comparison to expression regulated by the human elongation factor 1-α promoter. Ex vivo lentiviral transduction of an APro-regulated green fluorescent protein (GFP) construct in mouse marrow supported GFP expression throughout hematopoeitic lineages in primary transplant recipients and was sustained in secondary recipients. The human Artemis promoter thus provides sustained and moderate levels of gene expression that will be of significant utility for therapeutic gene transfer into hematopoeitic stem cells.
    DNA and cell biology 06/2011; 30(10):751-61. · 2.28 Impact Factor
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    ABSTRACT: Metastatic colon cancer is one of the leading causes of cancer-related death worldwide, with disease progression and metastatic spread being closely associated with angiogenesis. We investigated whether an antiangiogenic gene transfer approach using the Sleeping Beauty (SB) transposon system could be used to inhibit growth of colorectal tumors metastatic to the liver. Liver CT26 tumor-bearing mice were hydrodynamically injected with different doses of a plasmid containing a transposon encoding an angiostatin-endostatin fusion gene (Statin AE) along with varying amounts of SB transposase-encoding plasmid. Animals that were injected with a low dose (10 μg) of Statin AE transposon plasmid showed a significant decrease in tumor formation only when co-injected with SB transposase-encoding plasmid, while for animals injected with a higher dose (25 μg) of Statin AE transposon, co-injection of SB transposase-encoding plasmid did not significantly affect tumor load. For animals injected with 10 μg Statin AE transposon plasmid, the number of tumor nodules was inversely proportional to the amount of co-injected SB plasmid. Suppression of metastases was further evident in histological analyses, in which untreated animals showed higher levels of tumor cell proliferation and tumor vascularization than animals treated with low dose transposon plasmid. These results demonstrate that hepatic colorectal metastases can be reduced using antiangiogenic transposons, and provide evidence for the importance of the transposition process in mediating suppression of these tumors.
    Molecular Cancer 02/2011; 10(1):14. · 5.13 Impact Factor
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    ABSTRACT: Neuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture. Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP. The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.
    Molecular Pain 01/2010; 6:31. · 3.77 Impact Factor
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    ABSTRACT: Two methods of systemic gene delivery have been extensively explored, using the mouse as a model system: hydrodynamic delivery, wherein a DNA solution equivalent in volume to 10% of the mouse weight is injected intravenously in less than 10 sec, and condensation of DNA with polyethylenimine (PEI) for standard intravenous infusion. Our goal in this study was to evaluate quantitatively the kinetics of gene expression, using these two methods for delivery of Sleeping Beauty transposons. Transposons carrying a luciferase expression cassette were injected into mice either hydrodynamically or after condensation with PEI at a PEI nitrogen-to-DNA phosphate ratio of 7. Gene expression in the lungs and liver after hydrodynamic delivery resulted in exponential decay with a half-life of about 35-40 hr between days 1 and 14 postinjection. The decay kinetics of gene expression after PEI-mediated gene delivery were more complex; an initial decay rate of 6 hr was followed by a more gradual loss of activity. Consequently, the liver became the primary site of gene expression about 4 days after injection of PEI-DNA, and by 14 days expression in the liver was 10-fold higher than in the lung. Overall levels of gene expression 2 weeks postinjection were 100- to 1000-fold lower after PEI-mediated delivery compared with hydrodynamic injection. These results provide insight into the relative effectiveness and organ specificity of these two methods of nonviral gene delivery when coupled with the Sleeping Beauty transposon system.
    Human gene therapy 09/2009; 21(2):210-20. · 4.20 Impact Factor
  • Molecular Genetics and Metabolism - MOL GENET METAB. 01/2009; 96(2).
  • Molecular Genetics and Metabolism - MOL GENET METAB. 01/2008; 93(2):15-16.
  • Molecular Genetics and Metabolism - MOL GENET METAB. 01/2008; 93(2):23-23.
  • Molecular Genetics and Metabolism - MOL GENET METAB. 01/2008; 93(2):31-31.
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    ABSTRACT: Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTX-treated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vector-modified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.
    Journal of Pharmacology and Experimental Therapeutics 10/2007; 322(3):989-97. · 3.89 Impact Factor
  • Lalitha R Belur, Kelly Podetz-Pedersen, Joel Frandsen, R Scott McIvor
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    ABSTRACT: The Sleeping Beauty (SB) transposon is an integrative nonviral plasmid system. Here, we describe a protocol for SB-mediated transgene delivery using DNA/polyethyleneimine (PEI) complexes for long-term expression in mouse lungs. This protocol can be used for delivery of any plasmid-based vector system to mouse lungs, although long-term transgene expression will be obtained only when using the SB transposon or other integrating vector systems. The stages of this protocol are preparation of DNA-PEI complexes and injection of the complexes into the lateral tail vein of mice. We also provide protocols for assessing transgene expression using in vivo bioluminescence imaging and enzymatic assay of lung homogenates. The procedure can be completed within 24 h, starting from preparation of DNA-PEI complexes to analysis of transient transgene expression.
    Nature Protocol 02/2007; 2(12):3146-52. · 8.36 Impact Factor
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    ABSTRACT: Nonviral, DNA-mediated gene transfer is an alternative to viral delivery systems for expressing new genes in cells and tissues. The Sleeping Beauty (SB) transposon system combines the advantages of viruses and naked DNA molecules for gene therapy purposes; however, efficacious delivery of DNA molecules to animal tissues can still be problematic. Here we describe the hydrodynamic delivery procedure for the SB transposon system that allows efficient delivery to the liver in the mouse. The procedure involves rapid, high-pressure injection of a DNA solution into the tail vein. The overall procedure takes <1 h although the delivery into one mouse requires only a few seconds. Successful injections result in expression of the transgene in 5-40% of hepatocytes 1 d after injection. Several weeks after injection, transgene expression stabilizes at approximately 1% of the level at 24 h, presumably owing to integration of the transposons into chromosomes.
    Nature Protocol 01/2007; 2(12):3153-65. · 8.36 Impact Factor
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    ABSTRACT: Molecular Therapy (2006) 13, S70|[ndash]|S70; doi: 10.1016/j.ymthe.2006.08.205 181. Quantitative Evaluation of Long Term Gene Expression In Vivo after Delivery of Polyethylenimine-Conjugated Sleeping Beauty Transposon DNA Kelly M. Podetz-Pedersen1,3, Jason B. Bell1,2,3, Terry W. Steele4, Joel L. Frandsen1,3, Thomas W. Shier4, R. Scott McIvor1,3,5,|[ast]| and Perry B. Hackett1,2,3,|[ast]|1Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN2Gene Therapy Program, Department of Pediatrics, University of Minnesota, Minneapolis, MN3Beckman Center for Transposon Research and Institute of Human Genetics, University of Minnesota, Minneapolis, MN4Department of Pharmaceutics, University of Minnesota, Minneapolis, MN5Discovery Genomics, Inc., Minneapolis, MN|[ast]|R Scott McIvor and Perry Hackett has a financial interest in Discovery Genomics, Inc.
    Molecular Therapy 04/2006; · 7.04 Impact Factor