Jeffrey Nordstrom - Academia.edu (original) (raw)

Papers by Jeffrey Nordstrom

Steroids, 2003

Antiprogestin-controlled gene regulation systems, initially developed by Bert O'Malley and collea... more Antiprogestin-controlled gene regulation systems, initially developed by Bert O'Malley and colleagues, are based on the unusual properties of certain truncated progesterone receptor ligand-binding domains . These modified PR-LBDs have lost the ability to respond to progestins, but have gained the ability to respond to antiprogestins as agonists, rather than as antagonists. When a modified PR-LBD is joined to specific DNA-binding and transcription activator domains, the resultant chimeric protein functions as an antiprogestin-inducible transcription factor for transgenes linked to promoters with specific DNA-binding sites. Antiprogestin-inducible gene regulation systems have been used to regulate transgene expression in cultured cells, transgenic animals, and for in vivo gene transfer studies using viral-or plasmid-based vectors. We have designed a plasmid-based, muscle-specific GeneSwitch ® system that is delivered to skeletal muscle by electroporation and provides regulated erythropoietin (EPO) expression in mice and rats in a manner that is dependent on orally administered mifepristone (MFP). Regulation was effective at low doses of MFP and provided regulated biological responses (hematocrit changes) for more than 6 months. This plasmid-based, antiprogestin-inducible EPO/GeneSwitch ® system has the potential to be a convenient, long-lasting and effective gene-based therapy for the treatment of anemia.

Nucleic Acids Research, 1987

Transcripts originating from the SV40 late promoter of pSV2-neo or pSV2-cat contain pBR322 sequen... more Transcripts originating from the SV40 late promoter of pSV2-neo or pSV2-cat contain pBR322 sequences and are polyadenylated at the SV40 late poly(A) site, resulting in an RNA of 3500 nt. If the SV40(L) poly(A) signal is destroyed, late orientation transcripts are polyadenylated at a site within pBR322 sequences, yielding in an RNA of 2500 nt. This cryptic poly(A) site is located 42-46 nucleotides downstream from an AAUAAA. Utilization of the pBR322 poly(A) signal is undetectable in late orientation transcripts from pSV2-neo or pSV2-cat, although it is located 966 nucleotides upstream from the SV40(L) poly(A) signal. The pBR322 site is not utilized when the spacing between the two poly(A) signals is varied from 209 to 1913 nucleotides. The pBR322 poly(A) site was utilized only in constructs in which all or portions of the SV40(L) poly(A) signal were deleted, such as in a construct with a 7 bp deletion into the SV40(L) AATAAA and adjacent sequences.

Proceedings of The National Academy of Sciences, 1985

The region of pSV2neo that encompasses the simian virus 40 early polyadenylylation signal was rep... more The region of pSV2neo that encompasses the simian virus 40 early polyadenylylation signal was replaced with a DNA fragment that spans the 3' end of a sea urchin (Psammechinus miliaris) histone H2A gene. This clone, pMK2.H2A(3'), was used to transfect COS cells. RNA analysis revealed that transcripts from pMK2.H2A(3') were polyadenylylated at a site 85 nucleotides downstream from the expected 3' end of mature H2A mRNA. Nucleotide sequencing showed that the site of poly(A) addition was located 10 nucleotides downstream from a cluster of four A-A-U-A-A-A sequences.

Nucleic Acids Research, 1986

RNA mapping experiments and chloramphenicol acetyltransferase assays were used to analyze polyade... more RNA mapping experiments and chloramphenicol acetyltransferase assays were used to analyze polyadenylation in COS cells of transcripts from derivatives of pSV2-neo and pSV2-cat, in which the SV40 early poly(A) signal has been modified. Neither the sequence A-A-U-A-A-A nor the sequences located immediately downstream from it in the SV40 early gene appear to function by themselves as a poly(A) signal. When combined, however, these two elements form a poly(A) signal whose efficiency and specificity closely resemble those of the wild type signal. The addition of six nucleotides between the A-A-U-A-A-A sequence and the poly(A) site has no detectable effect on the efficiency or site of polyadenylation. Deletion of the 60 nucleotides immediately upstream from the hexanucleotide also has no detectable effect on polyadenylation. Therefore, A-A-U-A-A-A and sequences downstream from it appear to be sufficient for SV40 early polyadenylation.

Proceedings of The National Academy of Sciences, 1985

Biochemistry, 1982

Nuclear matrix was prepared by sequential treatment of oviduct nuclei with Triton X-100, DNase I,... more Nuclear matrix was prepared by sequential treatment of oviduct nuclei with Triton X-100, DNase I, and 2 M NaCl. Published procedures were modified such that as many steps as possible were performed at -20 OC to minimize endogenous ribonuclease activity. Examination of electron micrographs confirmed the isolation of intact nuclear matrix structures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins in these structures showed an absence of histones and an enrichment of certain nonhistone proteins. RNA was isolated from the nuclear matrix preparations and subjected to denaturing gel electrophoresis. Gels were analyzed by ethidium bromide staining and by hybridization of Northern blots to cloned DNA probes for ovalbumin, ovomucoid, 5.8s ribosomal RNA, and U1 RNA. All of the precursors to ovalbumin and ovomucoid mRNAs (including various splicing intermediates) and all of the precursors to Intervening sequences have been observed in nuclear RNA transcripts of animals, plants, and lower eucaryotes and in

Molecular Therapy, 2000

We investigated the ability of an improved mifepristone-dependent GeneSwitch system to regulate t... more We investigated the ability of an improved mifepristone-dependent GeneSwitch system to regulate the expression of genes for two therapeutic proteins: vascular endothelial growth factor (VEGF) and erythropoietin. The GeneSwitch system consisted of two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene. When the constitutive CMV promoter of the GeneSwitch plasmid was replaced by an autoinducible promoter consisting of four copies of GAL4 DNA binding sites linked to a minimal thymidine kinase promoter, the tightness of transgene regulation was improved by an order of magnitude. Quantitative RT-PCR analysis of GeneSwitch mRNA confirmed that the autoinducible promoter was responsive to mifepristone. We demonstrated the ability of the improved GeneSwitch system to regulate the expression of VEGF or erythropoietin in a biologically relevant manner after delivery of plasmids to the hind-limb muscle of adult mice. This ability of the autoinducible GeneSwitch system to regulate the expression of therapeutic proteins in mice indicates its potential for use in human gene therapy applications.

Gene Therapy, 1999

We have utilized a nonviral, polymeric interactive non-con-optimal doses of 24 and 48 g, respecti... more We have utilized a nonviral, polymeric interactive non-con-optimal doses of 24 and 48 g, respectively. While polydensing (PINC) gene delivery system to deliver IL-12 to morphonuclear cells (PMNs) were partially involved in the two different types of murine tumors, an immunogenic development of the antitumor immune response elicited by renal cell carcinoma, Renca, and a non-immunogenic IL-12/PVP treatment, CD8 + T cells were found to be the colon cell carcinoma, CT26. The delivery of IL-12/polyvinyl primary effectors. In contrast, CD4 + T cells did not appear pyrrolidone (PVP) complexes into Renca led to the to play a significant role in the development of tumor speexpression of IL-12 (146 ± 89 pg/mg) and IFN-␥ cific immunity. Finally, mice that rejected the tumors follow-(160 ± 82 pg/mg) from explanted tumors in culture super-ing IL-12/PVP treatment were protected against a second natants. Treated tumors showed increased infiltration of challenge with the same tumor. These data provide evi-NK, CD4 + and CD8 + T cells and up-regulation of MHC dence that a nonviral IL-12 gene delivery system is well class I molecules on leukocytes in both tumors and lymph tolerated and generates a potent immune response against nodes. Fifty per cent of tumor-bearing mice rejected Renca established tumors. or CT26 tumors following IL-12/PVP treatments given at

Human Gene Therapy, 1999

As gene therapy advances, the ability to regulate transgene expression will become paramount for ... more As gene therapy advances, the ability to regulate transgene expression will become paramount for safety and efficacy. In this study, we investigate the ability of the mifepristone-dependent GeneSwitch system to regulate the expression of trangenes delivered to mice by nonviral methods. Two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene for secreted human placental alkaline phosphatase (SEAP), were delivered to the hind-limb muscles of adult mice. Modulation of the level of secretion of the transgene product into serum was achieved by intraperitoneal administration of low doses of the drug mifepristone (MFP). The EC50 for induction of transgene expression by MFP was 0.03 +/- 0.005 mg/kg. The maximal level of transgene expression after induction was equal to or higher than that displayed by a plasmid driven by the CMV enhancer/promoter. The average magnitude of induction was 14- to 19-fold. Multiple rounds of drug-dependent regulation of transgene expression in vivo were demonstrated. In BALB/c mice, the ability to regulate transgene expression persisted for approximately 3 weeks, until the appearance of neutralizing antibodies to the secreted transgene product. In immune-deficient mice, the ability to repetitively regulate transgene expression persisted for at least 5 weeks. Although the dynamic range of regulation needs improvement, the plasmid-based GeneSwitch system has features that are attractive for gene therapy applications.

Human Gene Therapy, 1999

Administration of plasmid/lipid complexes to the lung airways for the treatment of metastatic pul... more Administration of plasmid/lipid complexes to the lung airways for the treatment of metastatic pulmonary diseases represents a new strategy of gene therapy. In this study we present evidence that intratracheal administration of a plasmid encoding murine IL-12 complexed with N-[1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride:cholesterol inhibits the growth of lung metastases, using a renal cell carcinoma model. Instillation of pIL-12/lipid complexes resulted in expression of biologically active IL-12 (170-240 pg/ml) and IFN-gamma (100-190 pg/ml) in the bronchoalveolar lavage fluid. A significantly reduced number of lung metastases (26+/-24) was observed in mice instilled with IL-12/lipid complexes 24 hr after tumor challenge, whereas more than 250 metastatic foci were counted in lungs of untreated mice. Moreover, IL-12/lipid inhibited the growth of 3-day-old established metastases when compared with empty plasmid/lipid or IL-12 plasmid in saline. Mice receiving IL-12 gene therapy survived significantly longer (median survival of 43 days) than untreated mice (median survival of 31 days) or mice treated with control plasmid/lipid complexes (median survival of 35 days). These data demonstrate that a nonviral IL-12 gene therapy employing synthetic cationic lipids as a delivery system can be used to inhibit the development of lung metastases. Thus, this method provides support for the use of IL-12/lipid complexes to control the growth of pulmonary metastases and represents a potentially safer alternative to IL-12 protein immunotherapy.

Expert Opinion on Biological Therapy, 2003

The ability to produce high-level transgene expression following the introduction of genetic mate... more The ability to produce high-level transgene expression following the introduction of genetic material into a host cell has been well documented. Various vectors and methods for in vivo gene delivery have been shown to provide long-term expression from many different tissue types in rodents and large animals. However, many potential therapeutic targets for gene therapy involve the production of proteins that are toxic or lead to undesirable effects if overexpressed. Thus, the ability to achieve regulated gene expression following treatment will be required to ensure the safety of long-acting gene therapy products. Skeletal muscle, in particular, has been widely used as a target for gene therapy protocols, due to the ease of accessibility and ability to produce and secrete some proteins at very high levels. This review focuses on regulated gene therapy systems that are being evaluated for use in muscle, and discusses two classes of system: those dependent on exogenously administered drugs and those dependent on endogenously produced metabolites.

Expert Opinion on Biological Therapy, 2003

Gene Therapy, 2002

injection of plasmids followed by electropermeabilization is an efficient process to deliver gene... more injection of plasmids followed by electropermeabilization is an efficient process to deliver genes into skeletal myofibers that permits proteins to be produced and secreted at therapeutically relevant levels. To further improve skeletal muscle as a bioreactor, we identified a formulation that elevates transgene expression in myofibers after i.m. injection and electroporation. With secreted placental alkaline phosphate (SEAP) as reporter gene, plasmid formulated with poly-L-glutamate produced two-to eight-fold higher levels of SEAP in mouse serum than plasmid in saline. Various concentrations and molecular weights of poly-L-glutamate were similarly effective, but 6 mg/ml of 15-50 kDa poly-L-glutamate consistently yielded the highest

Experimental Hematology, 2000

We have achieved therapeutic circulating levels of human Factor IX (hFIX) in mice and dogs using ... more We have achieved therapeutic circulating levels of human Factor IX (hFIX) in mice and dogs using plasmid-based gene delivery and electroporation. Plasmid encoding CMV-hFIX formulated with 5% polyvinylpyrrolidone (PVP) was injected into the tibialis and the gastrocnemius muscles of C57BLր6 mice, then electroporated using 1 cm 2 caliper electrodes. hFIX expression peaked at 144 ng mL Ϫ1 on day 7, but was undetectable at day 10. hFIX expression was dose-dependent. In SCID mice, plasma hFIX levels peaked at ‫021ف‬ ng mL Ϫ1 , were maintained for 3 months, then slowly diminished to ‫03ف‬ ng mL Ϫ1 at day 125. Redosing to the same muscle groups at day 153 injection increased hFIX expression levels to ‫09ف‬ ng mL Ϫ1 . In normal canines, multiple muscles of the rear and front legs were injected followed by electroporation with a six-needle array electrode. Peak mean levels of 36 ng mL Ϫ1 were achieved in the plasma of ‫01ف‬ kg dogs at 21 days, but was undetectable at day 28 due to antibody formation, confirmed by Western blot. Some animals showed transient increases (3-6x) in creatine kinase levels at day 3, indicating mild muscle trauma. Studies in dogs with hemophilia B are in progress. Plasmid-based gene delivery with a PINC TM polymer augmented with electroporation is scalable for large animals and represents a promising approach for prophylactic treatment of individuals with hemophilia B.

Experimental Hematology, 2000

mice were transduced by either DR.GFP or EF.GFP, and then cultured for 8 days to allow DC differe... more mice were transduced by either DR.GFP or EF.GFP, and then cultured for 8 days to allow DC differentiation. GFP expression was observed exclusively in the MHC II ϩ cell population derived from DR.GFP transduced BM cells, whereas transgene expression was observed equally in both MHC II ϩ and MHC II Ϫ cell populations derived from EF.GFP transduced BM cells. Thus, we have developed a gene transduction system to allow stable and specific transgene expression in APC, which has many applications for immunobiology and immunotherapy.

Steroids, 2003

Nucleic Acids Research, 1987

Proceedings of The National Academy of Sciences, 1985

Nucleic Acids Research, 1986

Proceedings of The National Academy of Sciences, 1985

Biochemistry, 1982

Molecular Therapy, 2000

Gene Therapy, 1999

Human Gene Therapy, 1999

Expert Opinion on Biological Therapy, 2003

Gene Therapy, 2002

Experimental Hematology, 2000