Identification and removal of colanic acid from plasmid DNA preparations: implications for gene therapy.

Polysaccharide contaminants in plasmid DNA, including current good manufacturing practices (cGMP) clinical preparations, must be removed to provide the greatest safety and efficacy for use in gene therapy and other clinical applications. We developed assays and methods for the detection and removal of these polysaccharides, our Super Clean DNA (SC-DNA) process, and have shown that these contaminants in plasmid DNA preparations are responsible for toxicity observed post-injection in animals. Furthermore, these contaminants limit the efficacy of low and high doses of plasmid DNA administered by numerous delivery routes. In particular, colanic acid (CA) that is mainly long-chained, branched and has high molecular weight (MW) is most refractory when complexed to cationic delivery vehicles and injected intravenously (IV). Because CA is often extremely large and tightly intertwined with DNA, it must be degraded, in order, to be effectively removed. We have produced a recombinant, truncated colanic acid degrading enzyme (CAE) that successfully accomplishes this task. Initially, we isolated a newly identified CAE from a bacteriophage that required truncation for proper folding while retaining its full enzymatic activity during production. Any plasmid DNA preparation can be digested with CAE and further purified, providing a critical advance to non-viral gene therapy.

A combinatorial approach for targeted delivery using small molecules and reversible masking to bypass nonspecific uptake in vivo.

We have developed a multi-disciplinary approach combining molecular biology, delivery technology, combinatorial chemistry and reversible masking to create improved systemic, targeted delivery of plasmid DNA while avoiding nonspecific uptake in vivo. We initially used a well-characterized model targeting the asialolglycoprotein receptor in the liver. Using our bilamellar invaginated vesicle (BIV) liposomal delivery system with reversible masking, we increased expression in the liver by 76-fold, nearly equaling expression in first-pass organs using non-targeted complexes, with no expression in other organs. The same technology was then applied to efficiently target delivery to a human tumor microenvironment model. We achieved efficient, targeted delivery by attachment of specific targeting ligands to the surface of our BIV complexes in conjunction with reversible masking to bypass nonspecific tissues and organs. We identified ligands that target a human tumor microenvironment created in vitro by co-culturing primary human endothelial cells with human lung or pancreatic cancer cells. The model was confirmed by increased expression of tumor endothelial phenotypes including CD31 and vascular endothelial growth factor-A, and prolonged survival of endothelial capillary-like structures. The co-cultures were used for high-throughput screening of a specialized small molecule library to identify ligands specific for human tumor-associated endothelial cells in vitro. We identified small molecules that enhanced the transfection efficiency of tumor-associated endothelial cells, but not normal human endothelial cells or cancer cells. Intravenous (i.v.) injection of our targeted, reversibly masked complexes into mice, bearing human pancreatic tumor and endothelial cells, specifically increased transfection to this tumor microenvironment approximately 200-fold. Efficacy studies using our optimized targeted delivery of a plasmid encoding thrombospondin-1 eliminated tumors completely after five i.v. injections administered once every week.

Selective expression of alternative lck mRNAs in human malignant cell lines.

The lck protein tyrosine kinase is normally expressed in a cell type-specific fashion, with mRNA being confined to cells of lymphoid lineage. Despite this highly specific pattern of expression in normal tissues, lck mRNA has also been detected in selected cell lines derived from human nonlymphoid neoplasms. In this study we explored the mechanisms underlying the expression of lck mRNA within human nonlymphoid neoplastic cell lines. We determined that lck mRNA expression was correlated with transcriptional activation and that there was no evidence for genomic rearrangement or amplification within the lck coding region to account for the expression of lck mRNA in the nonlymphoid neoplastic cell lines. The lck gene has previously been shown to contain two distinct promoter elements. In this study, we demonstrated that lck-producing cell lines derived from human nonlymphoid neoplasms expressed transcripts initiated exclusively from the 3'-most promoter element (3' promoter). In contrast, lymphoid cell lines derived from nonmalignant sources expressed lck transcripts exclusively initiated from the 5'-most promoter element (5' promoter). Most cell lines derived from human lymphoid neoplasms express lck transcripts initiated from both the 5' and 3' promoters in various ratios. Thus, lck expression in a variety of malignant cell lines results from a selective induction of transcription from the 3' promoter.

Improved DNA: liposome complexes for increased systemic delivery and gene expression.

To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.

Identification of a basal promoter for the human Mr 72,000 type IV collagenase gene and enhanced expression in a highly metastatic cell line.

A basal promoter for the Mr 72,000 type IV collagenase gene was specifically defined by chloramphenicol acetyltransferase assays of a nested set of 5' upstream fragments containing the promoter region. This core promoter is TACATCT and is a noncanonical TATA box that fits the TATA consensus sequence. This sequence begins 26 base pairs in the upstream direction from the start site of transcription for the type IV collagenase gene. This basal promoter is active in the highly metastatic A2058 melanoma cell line. A putative enhancer was found between nucleotides -223 to -422 that produces a 7-fold increase in transcriptional activity in the A2058 melanoma cell line. The region immediately 5' of the basal promoter, upstream to position -422, contains a silencer and represses transcriptional activity in the nonmetastatic HT144 melanoma cell line. The results of this study are consistent with previous data that found high expression of Mr 72,000 type IV collagenase mRNA and enzymatic activity in the A2058 cell line, whereas low mRNA expression and type IV collagenase activity were found in the HT144 cell line.

Cloning and characterization of human tumor cell interstitial collagenase.

A full-length complementary DNA (cDNA) for interstitial collagenase was isolated from an A2058 melanoma cDNA library using the pCD-X Okayama-Berg vector. The tumor interstitial collagenase cDNA was sequenced and compared to the published sequences for human fibroblast collagenase. The sequence for the tumor collagenase has two DNA base pairs which differ from the sequence of normal fibroblast collagenase. Restriction enzyme digestion of a specific DNA fragment produced by polymerase chain reaction amplification of genomic DNA from human placenta resolves a discrepancy in the previously reported DNA and amino acid sequences for the fibroblast collagenase. A high level of expression of interstitial collagenase message was found in human A2058 melanoma cells by Northern blot analysis, and this level was slightly increased by phorbol ester (phorbol myristate acetate) stimulation. Interstitial collagenase mRNA expression was significantly decreased by treatment with either transforming growth factor-beta 1 or retinoic acid in A2058 melanoma cells. A high level of the collagenase protein secreted into conditioned media was identified by Western blotting. As shown by gelatin zymogram analysis interstitial collagenase was one of at least two metalloproteinases secreted by this same cell line. Thus, human melanoma cells can directly produce interstitial collagenase without a requirement for host cell interaction.

Thrombospondin-1 promotes alpha3beta1 integrin-mediated adhesion and neurite-like outgrowth and inhibits proliferation of small cell lung carcinoma cells.

Although human small cell lung carcinoma (SCLC) cell lines are typically anchorage-independent and do not attach on most extracellular matrix proteins, OH-1, and several other SCLC cell lines attached on substrates coated with thrombospondin-1 (TSP1). SCLC cells grew long-term as adherent cells on a TSP1-coated substrate. Adhesion of SCLC cells on TSP1 was inhibited by heparin, function-blocking antibodies recognizing alpha3 or beta1 integrin subunits, and by soluble alpha3beta1 integrin ligands. SCLC cells extended neurite-like processes on a TSP1 substrate, which was also mediated by alpha3beta1 integrin. Process formation on a TSP1 substrate was specifically stimulated by epidermal growth factor and somatostatin. Adhesion on TSP1 weakly inhibited SCLC cell proliferation, but this inhibition was strongly enhanced in the presence of epidermal growth factor. TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 also inhibited proliferation when added in solution. High-affinity binding of 125I-labeled TSP1 to OH-1 cells was heparin-dependent and may be mediated by sulfated glycolipids, which are the major sulfated glycoconjugates synthesized by these cells. Synthesis or secretion of TSP1 by SCLC cells could not be detected. On the basis of these results, the alpha3beta1 integrin and sulfated glycolipids cooperate to mediate adhesion of SCLC cells on TSP1. Interaction with TSP1 through this integrin inhibits growth and induces neurotypic differentiation, which suggests that this response to TSP1 may be exploited to inhibit the progression of SCLC.

Characterization of hUCRBP (YY1, NF-E1, delta): a transcription factor that binds the regulatory regions of many viral and cellular genes.

The UCRBP (YY1, delta, NF-E1) protein has been isolated for its ability to bind to the UCR (upstream conserved region) site present in the conserved murine leukemia virus long terminal repeat. UCRBP carries a highly charged N-terminal domain and four C2-H2-type zinc fingers at its C-terminal end. The present study reveals the following results: (i) The UCR site is present in the upstream and/or regulatory regions of numerous mammalian cellular and viral genes to which both recombinant and cellular UCRBP bind. UCR sites are also found in the regulatory regions of repetitive sequences including human LINE-1 elements and mouse intracisternal-A particle sequences. (ii) By immunological and UV cross-linking experiments, we found that two proteins, of approx. 68 kDa and an antigenically related protein of approx. 40 kDa, account for much of the UCR-binding activity in T-lymphocytes. (iii) There is evidence that UCRBP acts as a phosphoprotein. Eight consensus phosphorylation sites are found in the deduced amino-acid sequence of human UCRBP. The cellular UCR-binding activity was abolished by phosphatase treatment, and there is an incremental increase in apparent molecular mass between the cytoplasmic and nuclear forms of the protein, suggesting phosphorylation. (iv) Although UCRBP has been previously shown to act as a transcriptional repressor, we show here that UCRBP can also act as a positive transactivator of a reporter driven by UCR elements when used in co-transfection assays. This transactivation occurred in a dose-restricted manner and was absent at high concentrations of a UCRBP expression plasmid, indicating a complex mode of function.(ABSTRACT TRUNCATED AT 250 WORDS)

Modeling human breast cancer metastasis in mice: maspin as a paradigm.

Breast cancer is the most common cancer detected in women, accounting for nearly one out of every three cancers diagnosed in the United States. Most cancer patients do not die from the primary tumor but die due to metastasis. Therefore, the study of metastasis is of most importance both to the clinician and patient. In the past, animal models have been used in breast cancer research and mammary gland biology. Our group has also established several animal models to address the function of a novel tumor suppressor gene maspin in breast tumor progression. Maspin was initially isolated from normal mammary epithelial cells. Its expression was down regulated in breast tumors. To test the protective role of maspin overexpression in mammary tumor progression, we crossed maspin overexpression transgenic mice (WAP-maspin) with a strain of oncogenic WAP-SV40 T antigen mice. The bitransgenic mice had reduced tumor growth rate and metastasis. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. In a separate animal experiment, maspin overexpressing mammary tumor cells (TM40D) were implanted into the fat pad of syngeneic mice. TM40D tumor cells were very invasive and metastatic. However, both primary tumor growth and metastasis were significantly blocked in TM40D cells that overexpress maspin as a consequence of plasmid or retrovirus infection. These evidences demonstrate that maspin function to inhibit primary tumor growth as well as invasion and metastasis. Elucidating the molecular mechanism of maspin action will shed light on our understanding of breast cancer invasion and metastasis.

The polymerase chain reaction. History, methods, and applications.

The polymerase chain reaction (PCR) uses in vitro enzymatic synthesis to amplify specific DNA sequences. PCR amplification can produce approximately 100 billion copies of one molecule of DNA in a few hours. PCR has revolutionized research in the biological sciences and medicine, and has influenced criminology and law. Several major scientific discoveries, including purification of DNA polymerase and elucidation of the mechanism of DNA replication, were essential for development of the present PCR technology. An overview of these discoveries and early work on in vitro DNA synthesis are presented. Basic PCR methodology, instrumentation, advanced PCR techniques, and applications are also discussed in this review. Several new amplification systems are mentioned. PCR is an extremely important and simple technology for research and diagnostic analyses of DNA and RNA. PCR technology and other amplification procedures will continue to produce novel applications in basic research and clinical medicine.

New directions in liposome gene delivery.

The history of liposomes, progress in liposome gene delivery, and future directions are discussed. Specific characteristics of liposomes and DNA:liposome complexes have been identified that are essential for optimal delivery and gene expression. Of particular interest are the requirements for increased delivery and high levels of gene expression in vivo. At present, significant efforts are focused towards achieving specific delivery and gene expression in target organs and tissues.

Intravenous delivery of liposome-mediated nonviral DNA is less toxic than intraperitoneal delivery in mice.

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells. Liposomes have been described as having better efficacy in gene delivery, and an advantage of using liposomes as gene carriers is that they can be used repeatedly in vivo. The objective of this study is to compare the effect of gene delivery routes and to determine whether systemic delivery of the rat insulin promoter (RIP)-directed suicide gene construct would permit cell-specific gene delivery in vivo. Severe combined immunodeficient (SCID) mice were injected with liposome-RIP-TK (thymidine kinase) complex by either the intraperitoneal or the intravenous route. Twenty-four hours post gene delivery, mice received ganciclovir (GCV) treatment twice daily for 14 days. Mice were sacrificed at various time points. Complete necropsy and serum chemistry analysis were performed. Islet morphology was determined using hematoxylin and eosin (H&E) staining. Serum glucose and insulin levels were also determined. To determine the toxic effect on pancreatic islet cells, immunostaining of insulin-producing and glucagon-producing cells was carried out at each time point. H&E staining indicated that both intravenous and intraperitoneal liposome-RIP-TK gene expression had no effect in normal endocrine islet cells. Both gene-delivery routes in mice resulted in normal glycemia and serum insulin levels. The endocrine islets were intact, with a normal distribution pattern of insulin-producing beta cells and glucagon-secreting alpha cells. However, serum chemistry analysis revealed significantly elevated levels of liver enzymes; suggesting that possible liver damage had occurred with the intraperitoneal gene delivery of liposome-pRIP-TK. Intravenous liposome-mediated gene delivery had no effect on liver enzyme levels. Liposome-mediated gene delivery via intravenous injection was less toxic than intraperitoneal delivery. This gene-delivery route requires fewer liposome-DNA complexes and maintains normal liver function. Thus, intravenous delivery of gene therapy would be superior to intraperitoneal administration of gene therapy in mice.

Complete foldback transposable elements encode a novel protein found in Drosophila melanogaster.

An apparently complete foldback (FB) transposable element homologous to FB white-crimson (FBwc) was analyzed. A complete FB element could encode one or more proteins required for regulation of FB transposition. The central DNA region (the loop) and the junctions between the loop and the inverted terminal repeats were sequenced. Three open reading frames (ORFs) are present in the loop, and a novel 308 bp inverted repeat is present at the junctions. No significant homologies were found when the DNA sequences of the loop region and the novel inverted repeat were screened against the Gene data bank. Antibodies were prepared in guinea-pigs against a peptide present near the amino terminus of ORF1, the longest ORF. A 71,000 dalton protein was isolated from an extract of Drosophila melanogaster early-stage embryos on an anti-ORF1 peptide-affinity column. Immunohistochemical studies of adult flies demonstrate localization of this protein in egg chambers.

Efficient gene targeting in mouse embryonic stem cells.

We developed methods to improve the efficiency of gene correction in mouse embryonic stem cells using homologous recombination of a replacement vector. The absolute frequency of homologous recombination in mouse embryonic stem (ES) cells, defined as the frequency of homologous recombination per electroporated cell, is approximately 10(-5) to 10(-6) by current procedures. Our method for gene targeting in mouse ES cells produces an absolute frequency of 10(-1). The protocol uses micro-electroporation chambers and a modified electroporation procedure that does not cause significant cell death. Plating and growth of the electroporated cells at an optimum density to maintain viability significantly increased the recovery of targeted cells. Due to the high frequency of targeting, corrected cells could be isolated by screening colonies obtained after growth without selection. Alternatively, colony formation and the absolute frequency could be increased by co-plating the electroporated cells with nonelectroporated ES cells before the addition of selective medium. These parental cells were nonirradiated but were killed in the selective medium. Plating density and efficiency of colony formation are therefore critical factors for obtaining a high absolute frequency of homologous recombination. Because this frequency is extremely high, these methods can be used to perform gene targeting without the use of selectable markers.

Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically.

The 20,500-dalton gamma delta resolvase monomer can be cleaved by chymotrypsin into a 5000-dalton COOH-terminal fragment and a 15,500-dalton NH2-terminal fragment that have been purified. Two crystal forms of the large fragment have been obtained, one of which is isomorphous with crystals of the native protein, showing that the large fragment makes the protein-protein contacts in the crystal and that the small fragment is segmentally disordered relative to the large fragment. Nuclease protection demonstrates that the small fragment binds specifically to all three DNA binding sites protected by resolvase. However, unlike native resolvase, which binds to all three complete sites with equal affinity, the small fragment binds to each of the six half sites with a different affinity. It has not been possible to demonstrate specific DNA binding of the larger fragment. Thus, resolvase has a modular construction analogous to that found for some repressors and activators; its COOH-terminal domain recognizes specific sequences in the DNA and its NH2-terminal domain mediates protein-protein interactions and probably has the enzymatic activity.

Insulinoma-induced hypoglycemic death in mice is prevented with beta cell-specific gene therapy.

OBJECTIVE AND SUMMARY BACKGROUND DATA: Tumor-specific gene therapy can be achieved if a tumor-specific promoter can be identified. In this study the authors investigated the use of the rat insulin promoter (RIP) for insulinoma-specific expression of a reporter gene. Insulinoma-specific cytotoxicity using the suicide gene thymidine kinase (tk) was studied both in vitro and in vivo. RIPtk gene therapy, delivered by a nontoxic, noninflammatory liposomal delivery system, was used in an insulinoma ICR/SCID mouse model to prevent hypoglycemic death. METHODS: Rat insulin promoter (0.502 kb) was ligated to the reporter gene lacZ and ligated to the tk gene. These two genes were transfected into a mouse insulinoma (NIT) cell line to ascertain insulinoma-specific expression and insulinoma-specific cytotoxicity in vitro. Reverse transcriptase-polymerase chain reaction and electrophoretic mobility-shift assays were performed on NIT-1 cell RNA and nuclear extract, respectively, to determine the transcription factors present and responsible for RIP activation in NIT-1 cells. A mouse insulinoma model was created with NIT-1 cells. These mice were treated with the RIPtk gene, and both blood sugars and animal viability were monitored. RESULTS: Only NIT-1 cells stained blue after X-gal staining or had detectable levels of beta-galactosidase protein. A significant decrease in cell survival was observed in NIT-1 cells transfected with RIPtk in vitro. Messenger RNA for both BETA2 and PDX-1 was found in NIT-1 cells, and a supershift was observed for both BETA2 and PDX-1. Experimental mice treated with the RIPtk gene, delivered by a liposomal gene delivery system, maintained their blood glucose levels, and the animals did not die of hypoglycemia. CONCLUSIONS: The data suggest that the RIP is an insulinoma-specific promoter. An ICR/SCID mouse insulinoma model was used to show that insulinoma-specific cytotoxicity can be accomplished by RIP coupled to a suicide gene in vivo, preventing hypoglycemic death.

Liposomal enhancement of the antitumor activity of conditionally replication-competent adenoviral plasmids.

Many human tumors have a functional deficiency in p53. Numerous studies have taken advantage of this phenomenon to use a conditionally replication-competent adenovirus (Ad dl1520) that will grow in and lyse tumor cells while sparing normal tissues. However, success has been limited, in part due to difficulties in reaching a sufficiently high proportion of tumor cells. Preexisting or developing immune responses directed toward viral proteins further decrease the efficacy of the approach. We have developed a liposome-encapsulated conditionally replication-competent plasmid based on the dl1520 virus. Like the parent virus, this plasmid generates infectious particles following transfection of p53-defective, but not p53-wild-type tumor cells, but unlike the parent virus it is able to infect CAR-negative tumor cells. The antitumor efficacy of this infectious plasmid was demonstrated in mice with xenografted human tumors, in which it was active after both local and intravenous administration for subcutaneous tumors and following intravenous administration for disseminated malignancy. Activity was retained systemically, even in the presence of neutralizing antibody. Such liposomally encapsulated conditionally replication-competent plasmids may complement the use of conventional viral particles, particularly in settings in which liver uptake of adenoviral vector is undesirable or there are problematic inhibitory effects from humoral immune responses.