HPV-16 E6 and E7 genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes. Therefore, the E6͞E7 genes can be considered relevant targets for anti-cancer therapy. We produced several engineered hairpin (HP) ribozymes to specifically disrupt HPV-16 E6͞E7 mRNA. After extensive biochemical characterization, one anti-E6 HP ribozyme (R434) was selected for in vivo testing because of its superior catalytic capabilities. When expressed in cis, R434 efficiently inhibited E6 in vitro translation. Cis-expression of the HP ribozyme with HPV-16 E6͞E7 genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6͞E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long term expression. Of interest, an inactive version of the HP also was able to significantly affect the immortalizing ability of E6͞E7, probably through passive hybridization. The combination of passive and cleaving antisense RNA therefore is established as an effective inhibitor of HPV-16 E6͞E7 immortalization.Over one-half of invasive cervical carcinomas worldwide and many cervical carcinoma-derived cell lines contain and express DNA from human papillomavirus type 16 (HPV-16). In early stages, HPV-16 expression causes benign proliferation and efficiently immortalizes cultured human epithelial cells, including cervical keratinocytes (1-3). The HPV-16 viral, early genes E6 and E7 are required to acquire and maintain efficiently the transformed phenotype in a broad spectrum of cell types and commonly are expressed in cervical carcinoma cells. Thus, E6͞E7 genes are the hallmark of cervical carcinoma (4-7). The E6 and E7 proteins bind with high affinity the p53 and Rb tumor suppressors, respectively (8, 9). The interaction of HPV-16 E6 protein with p53 results in degradation through the ubiquitin pathway, resulting in the equivalent of a mutant p53 phenotype (10-12). The association of E7 with Rb impedes the interaction of Rb with several proteins (i.e., E2F), efficiently disrupting the cell cycle (13-15). Therefore, the E6͞E7 genes are ideal targets for anti-cancer therapy.Antisense RNA and oligonucleotides have been used specifically to block translation of several genes. This effect is obtained by the hybridization of passive antisense molecules with their respective complementary mRNA to form nontranslatable double-stranded RNA molecules or DNA-RNA hybrids that promote the activity of endogenous RNase H, an enzyme that specifically digests the RNA strand of DNA-RNA hybrid molecules (16)(17)(18). In cultured tumor cells, antisense oligonucleotides have been shown to suppress effectively translation of several genes and to reverse some phenotypes (19)(20)(21)(22). However, passive antisense therapy has the disadvantage of being active for a limited period and often causing nonspecific toxicity (23,24). This approach has produced inhibition of E6͞E7 gene expression in HPV-18-containin...
Cell-penetrating peptides (CPPs) inhibit Herpes simplex virus entry at low micromolar concentrations and may be useful either as prophylactic or therapeutic agents for herpetic keratitis. The aim of this study was to assess the in vitro and in vivo toxicity of three CPPs-EB, TAT-C, and HOM (penetratin)-for the cornea. Incubation of primary (HK320) or immortalized (THK320) human keratocytes with the EB peptide (up to 100 microM), bHOMd (up to 200 microM), or TAT-C (up to 400 microM) resulted in no evidence of toxicity using a formazan dye-reduction assay. Similar results were obtained with a human trabecular meshwork cell line (TM-1), primary human foreskin fibroblasts (DP-9), Vero, and HeLa cells with EB and TATC. The bHOMd peptide showed some toxicity in Vero and HeLa cells, with CC50 values of 70 and 93 microM, respectively. The EB peptide did not inhibit macromolecular synthesis in Vero cells at concentrations below 150 microM, although cell proliferation was blocked at concentrations of EB above 50 microM. In vivo toxicity was assessed by applying peptides in Dulbecco's modified Eagle's medium to the cornea 4 times daily for 7 d. At concentrations 1000 times the IC50 values, the EB and bHOM peptides showed no toxicity, whereas TAT-C caused some mild eyelid swelling. Some slight epithelial cell sloughing was seen with the bKLA peptide in vivo. These results suggest that these CPPs-and EB in particular-have a favorable toxicity profile, and that further development is warranted.
Sequenom is an established genomic analysis company with two principal divisions: genetic analysis systems and reagents, and noninvasive prenatal diagnostics. As an industry innovator in DNA detection technology, Sequenom's benchtop MassARRAY Compact system allows a direct mass measurement of nucleic acids, providing unparalleled precision and quantitation. The MassARRAY system is a multi-application MALDI-TOF MS platform for genotyping, methylation and quantitative gene expression analysis. Sequenom continues to refine these core applications and has developed several new applications for copy number variation analysis and comparative sequence analysis for bacteria and viruses. On the diagnostics front, Sequenom is developing a comprehensive portfolio of prenatal test methods by combining the versatility of the MassARRAY system with the Company's SEQureDxtrade mark technology, which enables the detection of circulating cell-free fetal nucleic acids in a maternal blood sample.
These results suggest that mutant HSV lacking ribonucleotide reductase do not display oncolytic activity in the LHbetaTAg mouse and that this model may not be suitable for studying viral oncolysis as a therapy for retinoblastoma.
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