1. A Self-Processing Ribozyme Cassette: Utility against Human Papillomavirus 11 E6/E7 mRNA and Hepatitis B Virus 
Wei-Hua Pan, Ping Xin, John D Morrey, Gary A Clawson 
Molecular Therapy 
Volume 9, Issue 4, Pages (April 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
The SNIP cassette. (A) Diagrammatic representation of SNIP. The CLIP and CHOP portions of the SNIP cassette are shown beneath the upper diagram. Depicted is a double internal Rz (dITRz), and the various 3′ modifications (see text) are liberated with the dITRz. The sites of autocatalytic cleavage are designated S1–S4. The locations of the various primers used for QPCR amplification of regions of SNIP are indicated with arrows. (B) Primer combinations used for amplification of indicated regions of SNIP. The top row indicates the region of SNIP amplified. Primers used for RT are shown in the middle row, and primer pairs used for PCR amplifications are shown in the bottom row.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Autocatalytic processing and activity of SNIP cassettes. (A) In vitro autocatalytic processing of SNIP, SNIPHP, and SNIPHB cassettes. The SNIP cassettes, containing dITRz777/885 targeted to hepatitis B virus in both CLIP and CHOP portions, were used. In vitro transcription reactions were run for 5, 10, 20, or 60 min, after which reactions were terminated: These are designated 1–4, respectively. G shows a G-hydrolysis ladder after a 60-min transcription reaction containing [γ-32P]GTP. Reaction products were examined by PAGE on 8% sequencing gels, and the gels were dried and examined by autoradiography. Nucleotide sizes are shown to the left. Positions of the liberated dITRz from the SNIP cassette are indicated with the double arrows; dITRz differ slightly in size when liberated from the CLIP or CHOP portions of the cassette. As is evident, autocatalytic processing proceeds very efficiently in vitro. Similar results were also obtained with SNIPAA777/885 (data not shown), in which the liberated dITRz appear as slightly broader bands. (B) QPCR quantitation of SNIP cassette regions within cells. 293T cells were transfected with the various SNIP, SNIPHP, SNIPHB, or SNIPAA cassettes containing the 777/885 dITRz, and RNA was harvested 24 h later. Various primer pairs were used to amplify the respective regions of the cassettes (as described). Amplification regions are as indicated by the labels over the columns, and the version of the SNIP cassette is as designated to the left. Upper lines represent fluorescence for amplification cycles for the indicated SNIP regions, whereas the lower lines represent concurrent fluorescence for amplification cycles for 18S rRNA. For example, the upper left-hand box shows dITRz detected after transfections with the SNIPAA construct. The only portion of the various cassettes that was detectable was the liberated dITRz, although occasionally a very small relative amount of S2 and S4 regions was detectable, particularly with the SNIP cassette. (C) Effects of 3′ regions on stability of liberated dITRz within cells. 293T cells were transfected with the various SNIP cassettes containing the dITRz Rz777/885 in both CLIP and CHOP sites. 48 h later, RNA was harvested and quantitative RT/PCR was performed. Products were separated by PAGE on 8% gels and analyzed by autoradiography. Relative concentrations of the dITRz, compared with that from the SNIP cassette (no 3′-end modifications) were 2.6×, 2×, and 1.5× for the SNIPAA, SNIPHB, and SNIPHP cassettes, respectively. The same values were obtained using two primer pairs. Subsequent QPCR studies gave similar results, with relative concentrations of 2.2×, 1.5×, and 1.9×, respectively. The negative control sample had been transfected with an eGFP construct. (D) Effects of 3′-end modifications on catalytic activity in vitro. The dITRz Rz777/885 liberated from the various SNIP cassettes were tested in vitro with the corresponding HBV target RNA for 7, 20, or 60 min (as indicated) at 37°C in the presence of 5 mM MgCl2. Products were then separated by PAGE in 6% gels and the products analyzed by autoradiography. Catalytic activities relative to the dITRz liberated from the SNIP cassette are shown below. The catalytic activity of the dITRz liberated from the SNIPAA cassette was 130% of the control activity, dITRz activity from the SNIPHP cassette was the same as control activity, while that of the dITRz from the SNIPHB cassette was decreased by slightly more than 25%.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
mFold modeling of SNIPAA777/885. RNA was modeled using the RNA folding server found at Positions of the self-cleavage sites S1–S4 are shown. Color gradations indicate the propensity of nucleotides to appear in single-stranded conformations (red indicates that nucleotides are always found in single-stranded conformations in all minimum free-energy predictions, whereas black indicates that they are located in double-stranded conformations in all predictions).
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Identification of Rz cleavage sites in HPV11E6/E7 mRNA. Rz were designed based on the sites identified in the library selection procedures. Overlapping DNA oligonucleotides, which included an upstream T7 promoter, as previously described, were then synthesized [6,7]. Rz targeted to the sites were then transcribed in vitro and gel purified [7]. Rz (at 50 nM) were mixed with full-length 32P-labeled HPV11E6/E7 target mRNA (at 200 nM) and incubated for 1 h at 37°C, and the cut products were then examined by PAGE and autoradiography as described under Experimental Procedures. Rz409 was the most active Rz under these conditions. Detailed kinetic analyses were not performed here. However, the activity of Rz409 appeared similar to that of the HPV16 Rz targeted to the homologous region in HPV16E6/E7(Rz427), which showed a kcat/Km of 2.8 × 106 min−1 M−1 vs HPV16E6/E7 target, with a Km of approximately 50 nM.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
The effectiveness of the SNIPAARz expression cassette in cell culture. 293T human embryonal kidney cells were cotransfected with constructs containing HPV11E6/E7 target RNA and the various SNIPAARz constructs, containing either single ITRz or dITRz as indicated. At 1, 3, and 5 days following transfection, cellular RNA was isolated and examined using QPCR. All SNIPAARz constructs were effective at reducing target RNA levels, with the dITRz-containing constructs showing the greatest activity by day 5. Repeat experiments have shown analogous results, with 409/162 dITRz producing slightly greater reductions than 409/409 dITRz on day 5.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Effectiveness of the MIDGEAlb777/885 reagent. (A) Schematic view of the MIDGEAlb777/885 reagent. Hairpin oligonucleotides are ligated to the 5′/3′ ends of the Alb-SNIPAA777/885 construct, to produce a DNA construct without 5′ or 3′ ends. (B) Effects of MIDGEAlb777/885 in transgenic mice. MIDGEAlb777/885 or MIDGEAlbeGFP constructs (as indicated) were packaged in liposomes, targeted to hepatocytes using asialofetuin (ASF), and administered ip. In one group, MIDGEAlb777/885 was packaged in liposomes but not targeted with ASF. Seven days after the last dose, livers were removed (the interval was 10 days in the group treated once per week with MIDGEAlb777/885). From some tissue, DNA was extracted and HBV DNA was quantitated as described. Other samples were fixed in 10% buffered formalin and processed for immunohistochemistry for HBcAg as described. Results shown in the middle column represent log10 values as means ± standard errors, with statistical significance (***P < 0.001) as indicated. Results shown in the right column represent means ± standard errors of the fraction of hepatocytes in central venous regions showing cytoplasmic staining for HBcAg, with statistical significance as indicated (*P < 0.05).
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

8. Fig. 7
Immunohistochemical staining for HBV core antigen. Livers from transgenic animals were processed and stained for HBV core antigen as described. (A/B and C/D) Representative 10×/20× (original magnification) sections from two different representative animals treated twice per week for 2 weeks with the MIDGEAlb777/885 reagent and sacrificed 7 days after the final treatment. (E and F) Representative sections from two different representative untreated animals, and (G/H and I/J) 10×/20× (original magnification) sections from two different representative animals from the group treated once per week with the MIDGEAlb777/885 reagent and sacrificed 10 days later. In addition to loss of staining in nearly 90% of hepatocytes in the central venous regions, there is an obvious decrease in staining intensity within the residual immunoreactive hepatocytes. Bars denote 50 or 100 μm as indicated.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

9. Fig. 8
Immunohistochemical staining for HBV core antigen. Livers from transgenic animals were processed and stained for HBV core antigen as described. (A/B and C/D) 10×/20× (original magnification) photomicrographs from two different representative animals treated with the MIDGEAlb777/885 reagent using liposomes without ASF for targeting. (E/F) 10×/20× (original magnification) photomicrographs from a representative animal after treatment with the irrelevant MIDGEAlbeGFP construct. (G) A representative 10× (original magnification) photomicrograph from another untreated animal, and (H) a representative photomicrograph from another animal treated with the irrelevant MIDGEAlbeGFP construct. Bars denote 50 or 100 μm as indicated.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions