Xiaobing Tian

Targeted gene knockdown in zebrafish using negatively charged peptide nucleic acid mimics

Negatively charged homo‐oligomers of alternating trans‐4‐hydroxy‐L‐proline/phosphonate polyamides with DNA bases (HypNA‐pPNA) display excellent hybridization properties toward DNA and RNA, while preserving the mismatch discrimination, nuclease resistance, and protease resistance of peptide nucleic acids (PNAs). Similar properties are associated with morpholino phosphorodiamidate (MO) DNA mimics, which have been used in the model vertebrate zebrafish (Danio rerio) for genome‐wide, sequence‐based, reverse genetic screens during embryonic development. We evaluated mixed sequence HypNA‐pPNAs as an alternative to MOs, and found that even a single central DNA mismatch lowered the HypNA‐pPNA melting temperature by 16°C. We then observed that the melting temperatures of HypNA‐pPNA 18‐mers hybridized to RNA 25‐mers were comparable to the melting temperatures of MO 25‐mers, and that two HypNA‐pPNA mismatches lowered the melting temperature with RNA by 18°C. In zebrafish embryos we observed that HypNA‐pPNA 18‐mers displayed comparable potency to MO 25‐mers as knockdown agents against chordin, notail, and uroD, with greater mismatch stringency. Finally we observed that a specific HypNA‐pPNA 18‐mer elicited the dharma (bozozok)‐/‐ phenotype in zebrafish embryos, which MO 25‐mers do not. HypNA‐pPNAs designed to inhibit translation of specific zebrafish RNA targets thus demonstrated stringent hybridization properties, relative to DNA and MO oligomers, and present a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes. Developmental Dynamics, 2003.

PET Imaging of CCND1 mRNA in Human MCF7 Estrogen Receptor–Positive Breast Cancer Xenografts with Oncogene-Specific [64Cu]Chelator-Peptide Nucleic Acid-IGF1 Analog Radiohybridization Probes

Treatment of breast cancer is hampered by a large unmet need for rapid, sensitive, specific staging and stratification of palpable and nonpalpable abnormalities. Mammography and physical examination miss many early breast cancers, yet detect many benign lesions. Cyclin D1, encoded by CCND1 messenger RNA (mRNA), and insulin-like growth factor 1 receptor (IGF1R) are key regulators of cell proliferation that are overexpressed in most breast cancers. Therefore, we hypothesized that malignant breast masses could be imaged and quantitated externally by PET with a dual-specificity probe that targets both CCND1 mRNA and IGF1R. Methods: We designed a CCND1-specific peptide nucleic acid (PNA) hybridization sequence (CTGGTGTTCCAT), separated by a C-terminal spacer to a cyclized IGF1 peptide analog (d-Cys-Ser-Lys-Cys), for IGF1R-mediated endocytosis. On the N-terminus we attached a chelator (1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetyl [DO3A]) for the positron-emitting nuclide 64Cu. We administered the [64Cu]CCND1-IGF1 analog radiohybridization probes, as well as sequence controls, by tail vein to immunocompromised female NCr mice bearing human MCF7 estrogen-dependent, receptor-positive xenografts. We imaged the mice by PET and CT 4 and 24 h later, and measured tissue distribution of the radiohybridization probes. Results: We observed 8 ± 2-fold higher PET intensity in the center of the breast cancer xenografts than in the contralateral tissues at 24 h after injection of the [64Cu]CCND1-IGF1 analog radiohybridization probe. IGF1 blocking yielded significantly weaker images (P < 0.05) relative to the tumor-free side at 24 h after injection, as did a PNA mismatch probe, a peptide mismatch probe, and free 64CuCl2. Conclusion: These results are consistent with our hypothesis for radiohybridization PET of overexpressed CCND1 mRNA, dependent on IGF1R-mediated endocytosis, in suspect masses. Early noninvasive detection of initial cancerous transformation, as well as invasive or recurrent breast cancer, with dual-specificity radiohybridization probes, might enable molecularly targeted staging, stratification, and choice of therapy.

99mTc-peptide-peptide nucleic acid probes for imaging oncogene mRNAs in tumours.

Summary Imaging oncogene mRNA in tumours would provide a powerful tool for the early detection of occult malignant lesions. The goal was to prepare a chimera consisting of a dodecamer antisense peptide nucleic acid (PNA) specific for c‐MYC oncogene overexpressed in human breast cancer cells and a chelating moiety that facilitates quantitative radiolabelling with 99mTc and evaluate it for hybridization and tissue distribution in laboratory animals. The pentapeptide chelator‐PNA dodecamer specific for c‐MYC mRNA was extended from a solid support by 9‐fluorenylmethyloxycarbonyl (Fmoc) coupling. Similarly, a chelator‐PNA chimera with four central mismatches was also prepared which served as a control. The chimeras were purified, characterized and evaluated for hybridization to c‐MYC mRNA by fluorescent, real‐time polymerase chain reaction (RT‐PCR). The chimeras were labelled with 99mTc and their tissue distribution was examined in athymic nude mice bearing experimental human breast tumours. 99mTc radiolabelling was quantitative and presented a single peak in reversed phase liquid chromatography. Fluorescent real‐time polymerase chain reactions using primer and fluorescent probe sets previously calculated for c‐MYC mRNA demonstrated inhibition of reverse transcription by the c‐MYC specific chimera as compared to that of the control. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver, and appreciable levels in tumours. These observations suggest that 99mTc‐peptide‐PNA probes might be useful for imaging gene expression in tumours, and the approach is worthy of further investigation.

External imaging of CCND1, MYC, and KRAS oncogene mRNAs with tumor-targeted radionuclide-PNA-peptide chimeras.

Abstract: In 2005, breast cancer will kill approximately 40,410 women in the U.S., and pancreatic cancer will kill approximately 31,800 men and women in the U.S. Clinical examination and mammography, the currently accepted breast cancer screening methods, miss almost half of breast cancers in women younger than 40 years, approximately one‐quarter of cancers in women aged 40‐49 years, and one‐fifth of cancers in women over 50 years old. Pancreatic cancer progresses rapidly, with only 1% of patients surviving more than 5 years after diagnosis. However, if the disease is diagnosed when it is localized, the 5‐year survival is approximately 20%. It would be beneficial to detect breast cancer and pancreatic cancer at the earliest possible stage, when multimodal therapy with surgery, radiotherapy, and chemotherapy have the greatest chance of prolonging survival. Human estrogen receptor‐positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, elevated cyclin D1 protein due to overexpression of CCND1 mRNA, and elevated insulin‐like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC or CCND1 peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C‐terminus, and a Tc‐99m‐chelator peptide on the N‐terminus, could measure levels of MYC or CCND1 mRNA noninvasively in human IGF1R‐overexpressing MCF7 breast cancer xenografts in immunocompromised mice. Similarly, human pancreatic cancer cells typically display elevated levels of KRAS mRNA and elevated IGF1R. Hence, we also hypothesized that a KRAS Tc‐99m‐chelator PNA‐peptide probe could detect overexpression of KRAS mRNA in pancreatic cancer xenografts by scintigraphic imaging, or by positron emission tomography (PET) with a KRAS Cu‐64‐chelator PNA‐peptide. Human MCF7 breast cancer xenografts in immunocompromised mice were imaged scintigraphically 4‐24 h after tail‐vein administration of MYC or CCND1 Tc‐99m‐chelator PNA‐peptides, but not after administration of mismatch controls. Similarly, human Panc‐1 pancreatic cancer cells xenografts were imaged scintigraphically 4 and 24 h after tail‐vein administration of a KRAS Tc‐99m‐chelator PNA‐peptide, and AsPC1 xenografts were imaged by PET 4 and 24 h after tail‐vein adminstration of a KRAS Cu‐64‐chelator PNA‐peptide. The radioprobes distributed normally to the kidneys, livers, tumors, and other tissues. External molecular imaging of oncogene mRNAs in solid tumors with radiolabel‐PNA‐peptide chimeras might in the future provide additional genetic characterization of pre‐invasive and invasive breast cancers.

Imaging oncogene expression.

Abstract: In 2003, approximately 39,800 women in the US will die from breast cancer. Mammography and physical examination miss up to 40% of early breast cancers. Moreover, if an abnormality is found, an invasive diagnostic procedure must still be performed to determine if the breast contains atypia or cancer, even though approximately 85% of abnormalities are benign. Scintigraphic imaging of gene expression in vivo by noninvasive means could direct physicians to appropriate targets for intervention at the onset of disease and thereby significantly impact patient management. Until now, no method has been available to image specific overexpressed oncogene mRNAs in vivo by scintigraphic imaging. We hypothesize that gamma‐emitting Tc‐99m‐PNA‐peptides can be taken up by human ER+ and ER− breast cancer xenografts, hybridize to complementary mRNA targets in those cells, and concentrate sufficiently in tumor tissue to allow noninvasive imaging of oncogene overexpression. To prepare the probes, peptide analogs of insulin‐like growth factor 1 (IGF1) were extended from a solid support by Fmoc coupling. Peptide nucleic acid (PNA) dodecamers antisense to CCND1 and MYC mRNAs were then extended from the N‐terminus of IGF1, followed by a chelator peptide, using Fmoc coupling for all residues. The cysteine thiols were cyclized on the solid support, either before or after PNA extension. This simplified synthetic approach allows preparation of a variety of multipeptide disulfide‐bridged PNA chimeras. A chelating peptide‐PNA chimera antisense to MYC mRNA was then labeled efficiently with Tc‐99m, yielding a single product. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver and kidneys, with appreciable levels in tumors. This result enables testing of Tc‐99m‐peptide‐PNA probes to image gene expression in tumors.

Sequence specificity of alternating hydroyprolyl/phosphono peptide nucleic acids against zebrafish embryo mRNAs.

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)-/- phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.

Downregulation of Gene Expression with Negatively Charged Peptide Nucleic Acids (PNAs) in Zebrafish Embryos

We found that negatively charged, highly soluble PNA analogs with alternating phosphonates (HypNA-pPNAs) are effective and specific antisense agents in zebrafish embryos, showing comparable potency and greater specificity against chordin, ntl and uroD. In addition, we successfully phenocopied a dharma mutant that had not been found susceptible to MO knockdown. Both MO and HypNA-pPNAs against a tumor suppressor gene induced comparable upregulation of p53, illustrating similar effects on transcription profiles. HypNA-pPNAs are therefore a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes in zebrafish or validating newly identified orthologs, and perhaps for reverse genetic studies in other organisms.

Receptor-mediated internalization of chelator-PNA-peptide hybridization probes for radioimaging or magnetic resonance imaging of oncogene mRNAs in tumours

Early external detection of cancer gene activity might enable early treatment of cancer and might reduce cancer mortality. We hypothesized that oncogene mRNA overexpressed at thousands of copies per malignant cell in a zone of transformed cells could be imaged externally by scintigraphic imaging, PET (positron emission tomography) or MRI (magnetic resonance imaging) with PNA (peptide nucleic acid) hybridization probes that include chelators for metal cations and a cyclized peptide analogue of IGF-1 (insulin-like growth factor 1), D(Cys-Ser-Lys-Cys), to mediate internalization by IGF1R (IGF-1 receptor) overexpressed on cancer cells. We observed that human MCF7 breast cancer cells that overexpress IGF1R efficiently internalized fluorescein-chelator-PNA-D(Cys-Ser-Lys-Cys) to the cytoplasm, but not with D(Cys-Ala-Ala-Cys). Scintigraphic imaging of MCF7 xenografts in immunocompromised mice revealed that CCND1 and MYC [(99m)Tc]chelator-PNA-D(Cys-Ser-Lys-Cys) probes yielded xenograft. PET imaging with [(64)Cu]chelator-PNA-D(Cys-Ser-Lys-Cys) yielded stronger signals. Scintigraphic imaging of human AsPC1 pancreas cancer xenografts with [(99m)Tc]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys) yielded strong xenograft signals. Stronger xenograft image intensities were obtained by PET imaging of [(64)Cu]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys). MRI required extension of chelator-polydiamidopropanoate dendrimers from the N-termini of the PNA probes to increase the number of contrast paramagnetic gadolinium (III) cations per probe. These results provide a basis for detection of oncogene activity in tissues from outside the body by hybridization with metal-chelator-PNA-peptides that are selectively internalized by cancer cells.

TUMOR-TARGETING PEPTIDE-PNA-PEPTIDE CHIMERAS FOR IMAGING OVEREXPRESSED ONCOGENE mRNAS

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [99mTc]chelator-PNA-D(CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.

STa peptide analogs for probing guanylyl cyclase C

Guanylyl cyclase C (GC‐C), universally overexpressed on primary and metastatic colorectal carcinoma cells, is activated by endogenous ligands, guanylin, and uroguanylin, and by exogenous 18‐residue heat‐stable enterotoxins (STa) produced by diarrheagenic bacteria. Two 12‐residue STa analogs with alternate combinations of two interlocked disulfide bonds, peptides 3 and 6, were synthesized by orthogonal solid phase synthesis routes. Peptides 3 and 6 bound GC‐C with a rank order potency of STa > peptide 3 > peptide 6. Peptides 3 and 6 behaved as agonists in stimulating cGMP production. The results reveal that the toxic domain of STa can be reduced to 12 amino acids.