Characterization of a messenger RNA polynucleotide vaccine vector.

We have constructed mRNA transcripts encoding luciferase and human carcinoembryonic antigen (CEA) which are capped, polyadenylated, and stabilized by human beta-globin 5' and 3' untranslated regions. The mRNA construct encoding human CEA directed CEA expression in mouse fibroblasts in vitro following liposome-mediated transfection. The luciferase encoding mRNA transcripts mediated luciferase expression in vivo following i.m. injection. Based on the demonstration of protein expression in vitro and in vivo, the feasibility of using such a vector as a tumor vaccine was examined. In this pilot study, seven mice received 50 micrograms mRNA transcripts encoding CEA twice weekly for 5 weeks by i.m. injection followed by challenge with syngeneic, CEA-expressing tumor cells. This dose and schedule "primed" an immune response to CEA. Five of seven mRNA-immunized mice demonstrated anti-CEA antibody 3 weeks after tumor challenge whereas control mice had no evidence of antibody response. This strategy might be particularly useful to induce an immune response to a proto-oncogene product or growth factor which poses a risk of inducing malignant transformation consequent to prolonged protein expression.

Intracellular expression of a single-chain antibody directed against human papillomavirus type 16 E7 oncoprotein achieves targeted antineoplastic effects.

Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. Anti-HPV16 E7 intracellular single-chain antibodies (scFvs) were constructed to down-regulate HPV16 E7 oncoprotein in HPV DNA-containing cell lines. In these studies, we transfected anti-E7 scFvs into the HPV16-positive human cervical carcinoma cell lines CaSki and SiHa and tested them for their ability to inhibit cell proliferation and alter the level of HPV16 E7 oncoprotein. Our results showed that anti-HPV16 E7 scFvs inhibited cell proliferation by >85% in CaSki cells and by 95% in SiHa cells. E7 oncoprotein was down-regulated by anti-HPV16 E7 scFv, and its expression was inversely related to the amount of scFv transfected. However, there were no effects of transfecting scFvs alone in HPV-negative cell lines. These results imply that anti-HPV16 E7 scFvs only have specific anti-HPV16 E7 effects on cell proliferation and on the synthesis of virally encoded proteins in HPV-positive cell lines. Thus, transfection of HPV16 E7-positive tumors with antigen-specific scFvs may be a viable strategy for cervical cancer gene therapy.

Expression of human endogenous retrovirus k envelope transcripts in human breast cancer.

PURPOSE: We investigated the expression of human endogenous retroviral (HERV) sequences in breast cancer. EXPERIMENTAL DESIGN: Reverse transcription-PCR (RT-PCR) was used to examine expression of the envelope (env) region of ERV3, HERV-E4-1, and HERV-K in breast cancer cell lines, human breast tumor samples, adjacent uninvolved breast tissues, nonmalignant breast tissues, and placenta. Expression of HERV transcripts was confirmed by Northern blot analysis and in situ hybridization (ISH). To evaluate coding potential, amplified HERV sequences were cloned into vectors for expression and sequence analysis. RESULTS: No expression of ERV3 or HERV-E4-1 RNA was detected in the analyzed breast samples. In contrast, HERV-K transcripts were detected in most breast cancer cell lines and many breast tumor tissues. Expression was detected in a small percentage of matched, uninvolved breast tissues and in placentas but not nonmalignant breast tissues. In HERV-K-positive breast cancer tissues, Northern blot analysis demonstrated full-length proviral and spliced env transcripts. ISH demonstrated expression of HERV-K transcripts in breast tumor cells but not in normal or uninvolved breast epithelial cells. Independently isolated clones of HERV-K env cDNA generated recombinant proteins of the expected size. Sequence analysis of env cDNA clones derived from four breast tumor samples revealed >97% identity with the type I HERV-K102, with no premature termination codons. Independent isolates from the same breast tumor sample showed nucleotide sequence differences, suggesting that multiple loci may be transcribed. CONCLUSIONS: These data indicate that HERV-K transcripts with coding potential for the envelope region are expressed frequently in human breast cancer.

Phenotypic knock-out of the latent membrane protein 1 of Epstein-Barr virus by an intracellular single-chain antibody.

Epstein-Barr virus (EBV) causes lymphoproliferative diseases in immunocompromised patients and is associated with endemic Burkitt lymphoma, nasopharyngeal carcinoma and some cases of Hodgkin disease. The latent membrane protein 1 (LMP1) of EBV is a transmembrane protein that is essential for the transformation of B lymphocytes. LMP1-mediated up-regulation of Bcl-2 is thought to be an important element in this process. As an approach to explore novel treatments for EBV-associated lymphomas, we constructed a single-chain antibody (sFv) directed against LMP1 to achieve functional inhibition of this oncoprotein in EBV-transformed B lymphocytes. We demonstrated that intracellular expression of an endoplasmic reticulum (ER)-targeted form of this sFv markedly reduced LMP1 protein levels. We also observed a decrease in intracellular level of this protein which correlated with a marked reduction of Bcl-2 expression in EBV-transformed B lymphocytes. We further demonstrated that anti-LMP1 sFv-mediated reduction of Bcl-2 correlated with increased sensitivity of these cells to drug-induced cell death. Therefore, these data suggest that an anti-LMP1 sFv used in combination with conventional chemotherapy may be useful for gene therapy of EBV-associated lymphomas in immunocompromised patients.

A Sindbis virus mRNA polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo.

The direct intramuscular delivery of naked plasmid DNA has been demonstrated to allow expression of encoded heterologous genes in the target myocytes. The method has been employed to elicit immunization based upon delivery of antigen encoding plasmid DNA. For application in the context of achieving anti-tumor immunization against antigenic transforming oncoproteins, delivery of plasmid DNAs encoding these molecules would create significant potential safety hazards. As an alternative to DNA polynucleotide vectors, we explored the utility of mRNA vehicles for inducing foreign gene expression in muscle cells in vivo. Synthetic reporter-gene encoding mRNA transcripts were derived for this analysis. The Sindbis virus vector was also used to derive luciferase mRNA transcripts which possessed self-replication capacity. In these studies, it could be shown that the replicative vector was capable of directing significantly elevated levels of reporter gene expression in myocytes compared to a non-replicative mRNA species. In addition, the replicative species was capable of achieving significantly prolonged levels of in vivo gene expression compared to non-replicative mRNA. Both of these characteristics will make replicative mRNA vectors of utility for polynucleotide-based immunization protocols.

Functional knock-out of c-myb by an intracellular anti-c-Myb single-chain antibody.

Aberrant expression of the c-myb proto-oncogene is a key factor in the development of the neoplastic phenotype in a variety of contexts. On this basis, it has been proposed that ablation of c-myb function might be an effective approach for therapy. To this end, we have employed an intracellular single-chain antibody (sFv) approach to achieve the functional knock-out of the c-Myb onco-protein. We derived an anti-c-Myb sFv, which was configured into eukaryotic expression plasmids. We confirmed the expression of the cytoplasmic and nuclear forms of the sFvs in the correct subcellular compartments by immunofluorescent staining. Importantly, the anti-c-Myb sFvs strongly inhibited the transactivation activity of c-Myb. Furthermore, cytotoxic effect of the sFv was observed only in the c-Myb positive cell line K562. These results suggest that anti-c-Myb sFv is a valuable tool for understanding the molecular mechanisms of c-myb induced transformation. In addition, this approach may have potential utility in the gene therapy for c-myb-dependent malignant diseases.

Receptor-mediated gene delivery employing lectin-binding specificity.

Selective targeting of malignant cells will be necessary to implement many of the gene therapy strategies being designed to combat cancer. Targeting can be achieved by transductional or transcriptional approaches. Transductional targeting can be accomplished by exploiting differences in the molecules or receptors expressed on the cell surface of malignant versus normal cells. Given that malignant cells can be distinguished from normal by differences in the expression of cell surface carbohydrates, we hypothesized that transductional targeting would be feasible by molecular conjugate vectors which achieve cell binding by virtue of lectins directed against the cell surface glycocalyx. We have shown that gene transfer can be accomplished by these novel lectin-targeted molecular conjugate vectors and that lectin binding specificities may serve as a means for potential targeting of cancer cells for the purposes of gene therapy.

Omega-3 fatty acids decrease protein kinase expression in human breast cancer cells.

We report that 5-day exposure to physiological concentrations of eicosapentaenoic and docosahexaenoic acids resulted in a strong decrease in expression of the RIalpha regulatory subunit of protein kinase A and the PKC-alpha isozyme of protein kinase C in the human breast cancer cell line MDA-MB-231.