Jeffrey L. Nordstrom

Transcription of structural and intervening sequences in the ovalbumin gene and identification of potential ovalbumin mRNA precursors.

Structural sequences that are extensively separated by nonstructural intervening sequences in the natural ovalbumin gene are coordinately expressed in target and nontarget tissue. The intervening sequences, which consist of unique sequences in the chick genome, are transcribed in their entirety. The amount of nuclear RNA corresponding to these sequences, however, is approximately 10 times less than that observed for structural sequences. The accumulation of RNA corresponding to structural and intervening sequences during acute estrogen stimulation suggests either that there are different rates of transcription for these regions of the ovalbumin gene or that RNA sequences corresponding to the intervening sequences are preferentially processed and degraded. Comparison of the in vitro expression of portions of the ovalbumin gene in nuclei isolated from chronically stimulated oviducts indicates that both structural and intervening sequences are preferentially transcribed in vitro at rates approximately 500 times greater than expected for random transcription of the haploid chick genome. In addition, electrophoresis of oviduct nuclear RNA on agarose gels containing methylmercury hydroxide reveals multiple species of RNA that are from 1.3 to over 4 times larger than ovalbumin mRNA and hybridize to both structural and intervening sequences of the ovalbumin gene. These results are consistent with transcription of the entire ovalbumin gene into a large precursor molecule followed by excision of the intervening sequences and appropriate ligation of the structural sequences to form the mature mRNA.

Ligand-Dependent Regulation of Plasmid-Based Transgene Expression in Vivo

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.

Combination of Interleukin 12 and Interferon α Gene Therapy Induces a Synergistic Antitumor Response against Colon and Renal Cell Carcinoma

The antitumor effect and mechanism of action of IL-12 gene therapy combined with IFN-alpha gene therapy were investigated in tumor-bearing mice using renal and colon carcinoma models, Renca and CT26, respectively. Tumors were treated with murine IL-12 plasmid alone or in combination with IFN-alpha plasmid formulated with a polymeric interactive noncondensing (PINC) gene delivery system. Intratumoral injection of IL-12 DNA/polyvinyl pyrrolidone (PVP) alone induced rejection of 58 and 17% of Renca and CT26 tumors, respectively, whereas 25% (Renca) and 0% (CT26) rejection was observed in mice treated with IFN-alpha plasmid/PVP. Combination gene therapy of formulated plasmids, IL-12 with IFN-alpha, synergistically increased the antitumor response against Renca (100% tumor rejection) and CT26 (50%). In vivo depletion of leukocyte subsets indicated that CD8(+) T and NK cells were the primary effectors of the antitumor response induced by the combined cytokine gene therapy. Moreover, mice that rejected the primary tumors after combined treatment with IL-12 and IFN-alpha plasmid formulation developed protective immunity against a subsequent tumor challenge. Analysis of tumor-infiltrating leukocytes from mice treated with the combined IL-12 and IFN-alpha gene therapy showed upregulation of CD40 molecules on antigen-presenting cells (Mac-1(hi) cells). Finally, levels of mRNA for the chemokines IP-10 and TCA-3 were higher in tumors treated with the combination of cytokine plasmids than in tumors treated with either cytokine gene alone. These data provide evidence that IL12 gene therapy combined with IFN-alpha gene therapy synergistically induces regression of established tumors and may represent a novel therapeutic strategy for cancer treatment.

Processing of high molecular weight ovalbumin and ovomucoid precursor RNAs to messenger RNA

The existence of poly(A) sequences in the multiple high molecular weight forms of ovalbumin and ovomucoid nuclear RNA has been determined. The results indicate that all the bands observed in the total nuclear RNA including the largest (7.8 kb) were also detected in the poly(A) RNA. Kinetic labeling and chase experiments in oviduct tissue-suspension system indicated that the ovalbumin and ovomucoid high molecular weight RNAs which were labeled with a short-time incubation can be chased by cold nucleosides and actinomycin D into mature mRNAs. The largest RNAs labeled in this oviduct suspension system have a size of 7.8 kb for ovalbumin and 5.5 kb for ovomucoid, which correspond respectively to the a bands of steady state RNA. The processing of the precursors to mature mRNA was also examined by electron microscopic analysis, by hybridizing oviduct nuclear RNA to probes which were isolated from different segments of intervening DNA sequences and by measuring the turnover kinetics of specific intervening sequences present in cellular poly(A) RNA. The results indicate that the seven intervening sequence regions of the ovomucoid precursor are removed in a preferred but not necessarily an obligatory order.

“Blue heart”: characterization of a mifepristone-dependent system for conditional gene expression in genetically modified animals

A detailed characterization of a cardiac muscle-specific, ligand-regulated gene expression system was performed in transgenic mice using the inducing ligand mifepristone (MFP). Several lines of double transgenic mice were created that expressed a bacterial lacZ reporter gene in the heart, under the control of a MFP-activated transcription factor constitutively expressed in cardiac muscle. The transgenic mice, which were administered MFP at a dose of 1 micromol/l in the drinking water, responded to the ligand within 24 h. Induction of beta-galactosidase enzyme activity in the heart continued for up to 21 days and resulted in an average 17-fold increase in enzyme activity. The highest individual animal response measured was a 94-fold increase in enzyme activity. The EC(50) for MFP induction of beta-galactosidase activity in the heart was 0.7 micromol/l when MFP was administered in the drinking water. Pharmacokinetic analysis of MFP dosing in wild-type FVB/N mice showed that absorption was very rapid (T(max) 1-10 min), bioavailability was modest ( approximately 10%) and the t(1/2) of MFP in mouse plasma was determined to be approximately 5 h. Thus, the system functions effectively in transgenic mouse heart where induction of gene expression is sensitive and can be accomplished by a simple and broadly applicable drinking water protocol.

Regulated production of proteins from muscle using gene transfer: potential therapeutic applications

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.

The Ovalbumin Gene: Transcriptional Regulation by Estrogen

De novo synthesis of RNA sequences corresponding to intervening as well as to structural sequences of the ovalbumin gene have been detected in isolated oviduct nuclei. Their presence in the nuclear transcripts and their time course of induction support the hypothesis that transcription of structural and intervening sequences of the natural ovalbumin gene are regulated by steroid hormones. These results are in agreement with out previous demonstration of high-molecular-weight species of ovalbumin RNA in nuclei that contain structural as well as intervening RNA sequences and are thus likely precursors to mature cytoplasmic mRNAov. Analysis of the size of in vivo nuclear RNA by gel electrophoresis under denaturing conditions, revealed that withdrawal of hormone depletes the level of high molecular weight ovalbumin RNA as well as that of nature mRNAov and that readministration of estrogen induces the accumulation of both species. These results are consistent also with transcriptional regulation of the ovalbumin gene. In addition, they rule out the possibility that the rapid accumulation of mature mRNAov after secondard stimulation results from processing of ovalbumin RNA precursors that might have been stored in the withdrawn oviduct. We conclude that steroid hormones exert a primary effect at the level of gene transcription. Following this event, a series of coordinated cellular responses may occur which involve RNA processing, mRNA transport to the cytoplasm, protein synthesis and mRNA degradation. The final consequence of this network of molecular reactions is the induced cellular function inherent to a specific steroid hormone.