Breast cancer gene therapy using an adenovirus encoding human IL-2 under control of mammaglobin promoter/enhancer sequences.

Interleukin-2 (IL-2) has been used clinically for the treatment of some malignancies, but the toxicities associated with systemic IL-2 therapy are a major challenge. Here we have determined whether transcriptional targeting of IL-2 to breast cancer (BrCa) using an engineered human mammaglobin promoter/enhancer (MPE2) is a feasible option for reducing IL-2-associated toxicities while still achieving a meaningful antitumor effect. We have constructed nonreplicating adenovirus vectors encoding either a reporter gene (luciferase) or human IL-2 (hIL-2) complementary DNA under control of the MPE2 sequence, the murine cytomegalovirus immediate early (MCMV) promoter or the human telomerase reverse transcriptase (hTERT) promoter. Luciferase and hIL-2 complementary DNAs under the control of the MPE2 sequence in adenovirus vectors were expressed at high levels in BrCa cells and at lower levels in normal cells of human and murine origin. Cancer specificity of the hTERT promoter was found to be similar to that of the MPE2 promoter in cells of human origin, but reduced specificity in murine cells. The MPE2 regulatory sequence demonstrated excellent tissue specificity in a mouse tumor model. Whereas the MCMV promoter-controlled IL-2 vector generated high liver toxicity in mice, the MPE2-controlled IL-2 vector generated little or no liver toxicity. Both IL-2 vectors exerted significant tumor growth delay; however, attempts to further enhance antitumor activity of the IL-2 vectors by combining with the proapoptotic drug procaspase activating compound 1 (PAC1) were unsuccessful.

HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice.

Despite the tremendous potential of adenovirus (Ad) as a delivery vector for cancer gene therapy, its use in clinical settings has been limited, mainly as a result of the limited infectivity in many tumors and the wide tissue tropism associated with Ad. To modify the tropism of the virus, we have inserted the epidermal growth factor-like domain of the human heregulin-α (HRG) into the HI loop of Ad5 fiber. This insertion had no adverse effect on fiber trimerization nor did it affect incorporation of the modified fiber into infectious viral particles. Virions bearing modified fiber displayed growth characteristics and viral yields indistinguishable from those of wild-type (wt) virus. Most importantly, HRG-tagged virions showed enhanced infection of cells expressing the cognate receptors HER3/ErbB3 and HER4/ErbB4. This was significantly reduced in the presence of soluble HRG. Furthermore, HER3-expressing Chinese hamster ovary (CHO) cells were transduced by the HRG-modified virus, but not by wt virus. In contrast, CHO cells expressing the coxsackie-Ad receptor were transduced with both viruses. However, infection of an in vivo breast cancer xenograft model after intratumoral injection was similar with both viruses, suggesting that the tumor microenvironment and/or the route of delivery have important roles in infection of target cells with fiber-modified Ads.

Challenges and strategies for cystic fibrosis lung gene therapy.

Gene replacement therapy represents an interesting new approach for the treatment of cystic fibrosis (CF) lung disease. Basic research suggests that CF gene therapy is feasible, but major technological challenges must be addressed before clinical applications are likely to succeed. Therapeutic genes can be delivered to and expressed in human airways, but the number of cells expressing the transgene is relatively low. The inefficiency of gene delivery is largely attributable to the remarkable defenses of human airways. Maintaining long-term transgene expression in airway cells is also a significant obstacle. Recent advances have been made in the development of vectors, expression cassettes, and delivery techniques for enhancing airway gene transfer and expression. These advances have the potential to improve the efficiency of lung gene therapy and to achieve clinical benefits for CF patients in the future.

Gene vectors for cytokine expression in vivo.

The understanding of cytokine networks and the exploitation of these networks for the treatment of immune and inflammatory diseases as well as cancer depend on in vivo delivery of cytokines. Due to instability of recombinant cytokine proteins, investigators have employed cytokine-encoding gene therapy vectors to induce high levels of cytokine expression in vivo. Numerous gene therapy vectors have been developed recently which are suitable for this purpose. Recent advances in the design of adenovirus, adeno-associated virus, poxvirus, retrovirus, lentivirus, and nonviral vectors are described here. Properties of the various vector systems which determine their usefulness for cytokine gene delivery are compared. The implementations of cytokine-encoding gene therapy vectors for analyzing immune responses and for the therapy of inflammatory disorders, immune disease, infections and cancer are reviewed.

Regulation of allergic mucosal sensitization by interleukin-12 gene transfer to the airway.

Expression of granulocyte macrophage colony-stimulating factor (GM-CSF) in the airway allows allergic sensitization to ovalbumin (OVA) in an experimental protocol that others have shown to induce inhalation tolerance. The ensuing response is characterized by T helper (Th)2 cytokines, marked eosinophilia in the bronchoalveolar lavage fluid (BALF) and the tissue, and goblet-cell hyperplasia. These findings, which underscore the importance of the airway microenvironment in the development of immune responses to airborne antigens, prompted us to investigate whether a Type 1 polarized cytokine milieu in the airway would modulate the allergic sensitization. To this end, we concurrently expressed GM-CSF and interleukin (IL)-12 in the airway, using an adenovirus-mediated gene transfer approach. Coexpression of IL-12 did not prevent the development of an antigen-specific immune inflammatory response, but altered its phenotype. Whereas a similar total cell number was observed in the BALF, airway eosinophilia was abrogated. Histologic evaluation of the tissue corroborated the findings in the BALF and demonstrated that IL-12 coexpression prevented goblet-cell hyperplasia. Expression of IL-12 decreased IL-4 and IL-5 content in the BALF by about 80 and 95%, respectively, and IL-5 in the serum by approximately 80%. In contrast, interferon (IFN)-gamma was increased in both BALF and serum. Similarly, we observed a Th2/Th1 shift in OVA-specific cytokine production in vitro. Recall challenge with OVA in vivo after resolution of the initial inflammatory response demonstrated that the effect of IL-12 was persistent. IL-12-mediated inhibition of airway eosinophilia was mainly IFN-gamma-independent, whereas inhibition of OVA-specific IgE synthesis was IFN-gamma-dependent. Our data underscore the importance of the airway microenvironment in the elicitation of immune responses to environmental antigens.

Intratumoral coinjection of adenoviral vectors expressing IL-2 and IL-12 results in enhanced frequency of regression of injected and untreated distal tumors.

We have studied the ability of adenoviral (Ad) vectors expressing the cytokines IL-2 or IL-12 to mediate regression of established tumors in a mouse model of mammary adenocarcinoma. Previous results indicated that intratumoral injection of vectors expressing IL-2 (AdCAIL-2), or IL-12 (AdmIL-12.1) induced complete tumor regression in approximately 30-40% of treated animals. In the current studies, we investigated the mechanism of tumor killing in responding animals and the efficacy of AdIL-2 and AdIL-12 vector administration in combination compared with the use of either vector alone. Animals bearing subcutaneous mammary tumors were injected intratumorally with Ad vectors expressing IL-2 or IL-12 or were coinjected with both vectors. Animals receiving the combination treatment responded substantially better than animals which had received either vector alone, with 65% of animals treated with both vectors undergoing complete tumor regression. In all three treatment regimens, tumor regression was associated with the presence of specific antitumor antigen cytotoxic T-lymphocytes (CTLs), which secreted elevated levels of IFN-gamma. Consistent with circulating CTLs being involved in regression, when animals bearing bilateral tumors were inoculated in a single tumor with IL-2 or IL-12 expressing vectors, both tumors regressed in many cases. Again, treatment with both AdCAIL-2 and AdmIL-12.1 was most effective, with 63% of animals undergoing complete regression of both treated and untreated tumors, compared to 18 or 22% of animals injected with either AdCAIL-2 or AdmIL-12.1 alone. These data indicate that the combination of IL-2 and IL-12 is a more effective inducer of antitumor immune responses than either one alone, and that the resulting antitumor responses are effective in mediating the regression of distal untreated tumors, a property which may aid in the treatment of metastatic disease.

Adenovirus E1A under the control of heterologous promoters: wide variation in E1A expression levels has little effect on virus replication.

Adenovirus early region 1A(E1A) encodes a heterogeneous family of proteins some of which function as transactivators and are required for efficient viral replication in HeLa cells. We have constructed adenovirus type 5 (Ad 5) mutants in which the E1A transcription unit is placed under the control of either the human beta-actin promoter or the human cytomegalovirus immediate early promoter. The level of E1A expression in cells infected with these mutants was several times higher than that in wild-type virus infections. When the same heterologous promoters were inserted upstream of, but in the opposite orientation to, the E1A transcription unit, the level of expression was greatly reduced with respect to wild-type levels of E1A. Despite this variation of at least 40-fold in the concentration of E1A proteins in infected cells, there was no significant difference between wild-type Ad 5 and any of the mutants in their ability to replicate in HeLa cells. These results suggest that very low levels of E1A proteins are sufficient for virus production in cultured cells and that wild-type Ad 5 produces an amount of E1A in excess of that required.

Establishment of immortalized primate epithelial cells with sub-genomic EBV DNA.

The genetic information in a sub-fragment of EBV DNA, designated p31 (containing less than a quarter of the viral genome and derived from a recombinant DNA cosmid library) allows epithelial cells from primary monkey and human kidney cultures to escape senescence under standard tissue culture conditions. A number of epithelial cell lines, designated M1/31, 483/31, 199/31 and HK/31, have been established and characterized following transfection of primary cells with p31 DNA. They share many properties, although morphologically they are not all identical. The cultures are immortalized but not fully transformed or tumorigenic. They appear to be phenotypically stable, although DNA hybridization studies indicate that genotypic alterations, including amplification, occur subsequent to transfection with p31 DNA and the establishment of a continuously proliferating epithelium. All cell lines consistently express high levels of cytokeratin 18 and varying amounts of cytokeratin 7, demonstrating their epithelial origin. From a single marmoset kidney (designated 199) a series of related immortalized cells, with subtle phenotypic differences, have been generated by p31 or sub-fragments of it. Although hallmarks of a "hit-and-run" mechanism are apparent in all of our studies, 2 different techniques (in situ hybridization or selection for cell survival in semi-solid media, followed by nucleic acid hybridization) show that, in late-passaged cultures, a small proportion of the cells still contain some viral DNA. The studies focus on genetic information within the BamHI A and I regions as being relevant to immortalization. The role of the EBV DNA fragment in the genesis of epithelial cell lines is considered.

EBV gene expression in an NPC-related tumour.

A nasopharyngeal carcinoma tumour (designated C15) propagated in nude mice has been used to generate a large cDNA library that we have analysed for Epstein-Barr virus (EBV) gene expression. No gross alterations exist in viral DNA from C15 relative to other human isolates and the large deletion present in the B95-8 'prototype' viral strain established in marmoset cells is not found; C15 contains no linear virion DNA. In the cDNA library, of the six EBV nuclear antigens (EBNAs) expressed in latently infected B-lymphocytes, only clones for EBNA-1 are found. These data are confirmed by immunoblotting. Sequence analysis shows the EBNA-1 mRNA splicing pattern in the carcinoma to differ from that observed in B-lymphocytes. Further, contrary to observations with B-cell lines, most viral transcription in the tumour is localized onto the 'rightmost' region of the conventional EBV physical map. Transcripts identified corresponding to known genes include those for the latent membrane protein (LMP), the alkaline DNA exonuclease and probably the terminal protein; major transcripts are also derived from the BamHI D fragment and the region deleted in B95-8 EBV DNA. Novel transcripts have also been identified that proceed in an anti-sense direction to genes encoding functions associated with replication, such as the viral DNA polymerase. They contain a large, hitherto unidentified, open reading frame in the viral genome that is complementary to the putative function known as BALF3 and a smaller open reading frame complementary to BALF5 (the DNA polymerase gene). From the present studies we can conclude that: (i) EBV transcription patterns in the epithelial cells vary markedly from those identified previously in B-cells, reflecting differential use of promoters or splicing patterns. (ii) Transcription is tightly regulated and restricted in the C15 tumour with many latent genes, notably EBNAs 2-6, being 'switched off.' (iii) A family of cytoplasmic RNAs are transcribed in an antisense direction to a number of existing open reading frames in the EBV genome. (iv) There are a number of mutations in C15 transcripts relative to the B95-8 genome, some of which could result in amino acid alterations in proteins.

De novo and maintenance DNA methylation by a mouse plasmacytoma cell DNA methyltransferase.

A DNA methyltransferase of Mr = 140,000 that is active on both unmethylated and hemimethylated DNA substrates has been purified from the murine plasma-cytoma cell line MPC 11. The maximal rate of methylation was obtained with maintenance methylation of hemimethylated Micrococcus luteus or M13 DNAs. At low enzyme concentrations, the highest rate of de novo methylation occurred with single-stranded DNA or relatively short duplex DNA containing single-stranded regions. Strong substrate inhibition was observed with hemimethylated but not unmethylated DNA substrates. Fully methylated single-stranded M13 phage DNA inhibited neither the de novo nor the maintenance reactions, but unmethylated single-stranded M13 DNA strongly inhibited the maintenance reaction. The kinetics observed with hemimethylated and single-stranded substrates could be explained if the enzyme were to bind irreversibly to a DNA molecule and to aggregate if present in molar excess. Such aggregates would be required for activity upon hemimethylated but not single-stranded DNA. For de novo methylation of duplex DNA, single-stranded regions or large amounts of methyltransferase appear to be required. The relative substrate preference for the enzyme is hemimethylated DNA greater than fully or partially single-stranded DNA greater than fully duplex DNA.

Cell aggregate suspension culture for large-scale production of biomolecules.

A method is described for the rapid reversible conversion of a number of continuous cell lines from anchorage-dependent growth to growth as aggregates of cells in suspension culture. Employing this technique, an inoculum of three 75-cm2 flasks of BALB/c SV3T3 cells was grown to 60 liters of aggregate suspension in 14 days. This yielded 120 ml of packed cells or 9.1 x 10(10) cells. Similar results were obtained for other cell lines. Biomolecules such as migration-inhibition factor (MIF) and plasminogen activator were produced from these cultures.