In vitro cleavage of HPV16 E6 and E7 RNA fragments by synthetic ribozymes and transcribed ribozymes from RNA‐trimming plasmids

He, Yu; Lu, Chang-De; Qi, Guo

doi:10.1016/0014-5793(93)81102-6

Cited by 18 publications

(16 citation statements)

References 13 publications

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“…[9][10][11][12] Hammerhead ribozymes, for instance, have been used to cleave RNA in transgenic plants and animals. [13][14][15][16][17][18] Although it is clear that hammerhead ribozymes can cleave specific viral RNA or mRNA in cell-free systems effectively, the efficiency of cleavage in vivo remains to be improved.…”

Section: Introductionmentioning

confidence: 99%

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

et al. 2003

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The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5 0 /3 0 ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems -exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5 0 /3 0 ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.

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Section: Introductionmentioning

confidence: 99%

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

et al. 2003

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“…HH ribozymes directed against E6͞E7 genes have resulted in specific processing of HPV-16 and HPV-18 targets (36)(37)(38)(39). However, only HH ribozymes directed against a HPV-18 target have been shown to inhibit growth of cultured tumor cells to some degree (39).…”

mentioning

confidence: 99%

Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

Álvarez-Salas

Cullinan²,

Siwkowski

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Ribozymes co-transfected into HepG2 cells revealed that HBV RNA levels decreased in a dosedependent manner due to antisense and catalytic activity. Ribozymes also significantly inhibited HBV (McHutchison & Patel 2002 ID: 34630)replication [67].…”

Section: Anti-poly(a) Hbv Ribozymementioning

confidence: 98%

“…First, two HH ribozymes were developed and targeted to the E6 and E7 protein coding regions of HPV type 16 (HPV-16) genome (nt 240 and 597, respectively) [67]. The ribozymes were tested against target sequences with different lengths showing that target size is important for catalytic activity.…”

Section: Anti-hpv Ribozymesmentioning

confidence: 99%

Utilization of Ribozymes as Antiviral Agents

Benítez‐Hess

Álvarez-Salas

2006

LDDD

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Ribozymes are involved in strategies to block gene expression and have been used as agents against viral infections and cancer. Emphasis has been on studies using hairpin and hammerhead ribozymes. The uniqueness of ribozymes is that they posses multiple turnover allowing high efficiency as antiviral agents. Ribozymes have been directed to human immunodeficiency virus (HIV), hepatitis (B, C and E) and human papillomavirus (HPV), the cause of cervical cancer. The mode of action of antiviral ribozymes, their structure and their in vitro effectiveness as inhibitors of HIV, HBV, HCV, HEV and HPV is reviewed. Although ribozymes have been demonstrated as effective antivirals in cell culture and some animal models, clinical studies have been limited. Nevertheless, ribozymes remain as a viable alternative to antisense and RNA interference.

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In vitro cleavage of HPV16 E6 and E7 RNA fragments by synthetic ribozymes and transcribed ribozymes from RNA‐trimming plasmids

Cited by 18 publications

References 13 publications

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

Utilization of Ribozymes as Antiviral Agents

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