Wei-Hua Pan

Antisense applications for biological control

Although Natures antisense approaches are clearly impressive, this Perspectives article focuses on the experimental uses of antisense reagents (ASRs) for control of biological processes. ASRs comprise antisense oligonucleotides (ASOs), and their catalytically active counterparts ribozymes and DNAzymes, as well as small interfering RNAs (siRNAs). ASOs and ribozymes/DNAzymes target RNA molecules on the basis of Watson‐Crick base pairing in sequence‐specific manner. ASOs generally result in destruction of the target RNA by RNase‐H mediated mechanisms, although they may also sterically block translation, also resulting in loss of protein production. Ribozymes and DNAzymes cleave target RNAs after base pairing via their antisense flanking arms. siRNAs, which contain both sense and antisense regions from a target RNA, can mediate target RNA destruction via RNAi and the RISC, although they can also function at the transcriptional level. A considerable number of ASRs (mostly ASOs) have progressed into clinical trials, although most have relatively long histories in Phase I/II settings. Clinical trial results are surprisingly difficult to find, although few ASRs appear to have yet established efficacy in Phase III levels. Evolution of ASRs has included: (a) Modifications to ASOs to render them nuclease resistant, with analogous modifications to siRNAs being developed; and (b) Development of strategies to select optimal sites for targeting. Perhaps the biggest barrier to effective therapies with ASRs is the “Delivery Problem.” Various liposomal vehicles have been used for systemic delivery with some success, and recent modifications appear to enhance systemic delivery, at least to liver. Various nanoparticle formulations are now being developed which may also enhance delivery. Going forward, topical applications of ASRs would seem to have the best chances for success. In summary, modifications to ASRs to enhance stability, improve targeting, and incremental improvements in delivery vehicles continue to make ASRs attractive as molecular therapeutics, but their advance toward the bedside has been agonizingly slow. J. Cell. Biochem. 98: 14–35, 2006.

Systemic Targeting Inhibitor of κB Kinase Inhibits Melanoma Tumor Growth

Constitutive activation of nuclear factor-kappaB (NF-kappaB) has been directly implicated in tumorigenesis of various cancer types, including melanoma. Inhibitor of kappaB kinase (IKK) functions as a major mediator of NF-kappaB activation. Thus, development of an IKK-specific inhibitor has been a high priority, although it remains unclear whether systemic inhibition of IKK will provide therapeutic benefit. In this study, we show that inhibition of NF-kappaB activity in melanocytes that are persistently expressing an active H-Ras(V12) gene and are deficient in the tumor suppressors inhibitor A of cyclin-dependent kinase 4/alternative reading frame results in reduction of melanoma tumor growth in vivo. This effect is, at least in part, via regulation of NF-kappaB nuclear activation and RelA phosphorylation. Based on this result, we developed a double hammerhead ribozyme long-term expression system to silence either IKKalpha or IKKbeta. The ribozymes were placed in an EBV construct and delivered i.v. to nude mice bearing melanoma lesions, which developed after i.v. injection of H-Ras-transformed melanoma cells. Our in vivo data show that knockdown of endogenous IKKbeta significantly reduces the growth of the melanoma lesions and knockdown of either IKKalpha or IKKbeta prolongs the life span of immunocompetent mice.

A selection system for identifying accessible sites in target RNAs.

Although ribozymes offer tremendous potential for posttranscriptionally controlling expression of targeted genes, their utility is often limited by the accessibility of the targeted regions within the RNA transcripts. Here we describe a method that identifies RNA regions that are accessible to oligonucleotides. Based on this selection protocol, we show that construction of hammerhead ribozymes targeted to the identified regions results in catalytic activities that are consistently and substantially greater than those of ribozymes designed on the basis of computer modeling. Identification of accessible sites should also be widely applicable to design of antisense oligonucleotides and DNAzymes.

Intracellular localization of the tumor suppressor HtrA1/Prss11 and its association with HPV16 E6 and E7 proteins.

We have a long‐standing interest in a nuclear protease which appears to be involved in carcinogenesis. We recently identified the protease as high temperature requirement factor A 1 (HtrA1), also known as Prss11, which is member of an oxidative stress‐response family of proteases. HtrA1 has been classified as a secreted protease involved in TGFβ signaling, but recent work has shown HtrA1 to be a tumor suppressor. Here we show that processed forms of HtrA1 are found intracellularly and intranuclearly, and the active intranuclear form of HtrA1 shows an ∼Mr 29,000. Further, expression of HPV E6/E7 proteins is associated with a post‐transcriptional up‐regulation of HtrA1 (most notably the nuclear form), and HtrA1 is found associated with both HPV E6 and E7 proteins. J. Cell. Biochem. 105: 81–88, 2008.

Rapid Identification of Efficient Target Cleavage Sites Using a Hammerhead Ribozyme Library in an Iterative Manner

A major limitation to the effectiveness of ribozymes is definition of accessible sites in targeted RNAs. Although library selection procedures have been developed, they are generally difficult to perform and have not been widely employed. Here we describe a selection technology that utilizes a randomized, active hammerhead ribozyme (Rz) library in an iterative manner. After two rounds of binding under inactive conditions, the selected, active Rz library is incubated with target RNA, and the sites of cleavage are identified on sequencing gels. We performed this library-selection protocol using human papillomavirus type 16 E6/E7 mRNA as target and constructed Rz targeted to the identified sites. Rz targeted to sites identified with this procedure were generally highly active in vitro and, more importantly, they were highly active in cell culture, whereas their catalytically inactive counterparts were not. This protocol can be used to identify a set of potential target sites within a relatively short time.

Nanoresonator Chip-Based RNA Sensor Strategy for Detection of Circulating Tumor Cells: Response using PCA3 as a Prostate Cancer Marker

There is widespread interest in circulating tumor cells (CTCs) in blood. Direct detection of CTCs (often < 1/mL) is complicated by a number of factors, but the presence of ∼10(3) to 10(4) copies of target RNA per CTC, coupled with simple enrichments, can greatly increase detection capability. In this study we used resonance frequency shifts induced by mass-amplifying gold nanoparticles to detect a hybridization sandwich bound to functionalized nanowires. We selected PCA3 RNA as a marker for prostate cancer, optimized antisense binding sites, and defined conditions allowing single nucleotide mismatch discrimination, and used a hybrid resonator integration scheme, which combines elements of top-down fabrication with strengths of bottom-up fabrication, with a view to enable multiplexed sensing. Bound mass calculated from frequency shifts matched mass estimated by counting gold nanoparticles. This represents the first demonstration of use of such nanoresonators, which show promise of both excellent specificity and quantitative sensitivity.

Down-Regulation of HtrA1 Activates the Epithelial-Mesenchymal Transition and ATM DNA Damage Response Pathways

Expression of the serine protease HtrA1 is decreased or abrogated in a variety of human primary cancers, and higher levels of HtrA1 expression are directly related to better response to chemotherapeutics. However, the precise mechanisms leading to HtrA1 down regulation during malignant transformation are unclear. To investigate HtrA1 gene regulation in breast cancer, we characterized expression in primary breast tissues and seven human breast epithelial cell lines, including two non-tumorigenic cell lines. In human breast tissues, HtrA1 expression was prominent in normal ductal glands. In DCIS and in invasive cancers, HtrA1 expression was greatly reduced or lost entirely. HtrA1 staining was also reduced in all of the human breast cancer cell lines, compared with the normal tissue and non-tumorigenic cell line controls. Loss of HtrA1 gene expression was attributable primarily to epigenetic silencing mechanisms, with different mechanisms operative in the various cell lines. To mechanistically examine the functional consequences of HtrA1 loss, we stably reduced and/or overexpressed HtrA1 in the non-tumorigenic MCF10A cell line. Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers. A concomitant decrease in expression of epithelial biomarkers and all microRNA 200 family members was also observed. Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation. Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways.

The shorter the better: reducing fixed primer regions of oligonucleotide libraries for aptamer selection.

Oligonucleotide aptamers are highly structured DNA or RNA molecules, or modified versions thereof, that can bind to targets with specific affinities comparable to antibodies. They are identified through an in vitro selection process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment) to recognize a wide variety of targets, from small molecules to proteins, and from cultured cells to whole organisms. Aptamers possess a number of desirable properties, such as ease of synthesis, stability, robustness, and lack of immunogenicity. Standard SELEX libraries require two primers, one on each side of a central random domain, to amplify the target-bound sequences via PCR or RT-PCR. However, these primer sequences cause non-specific binding by their nature, and have been reported to lead to large numbers of false-positive binding sequences, or to interfere with binding of sequences within the random regions. This review is focused on methods which have been developed to eliminate fixed primer interference during the SELEX process.

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies (1). They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules (2-4). Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues. Aptamers that are identified through the standard SELEX process usually comprise approximately 80 nucleotides (nt), since they are typically selected from nucleic acid libraries with approximately 40 nt long randomized regions plus fixed primer sites of approximately 20 nt on each side. The fixed primer sequences thus can comprise nearly approximately 50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process (3), although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection (5). To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX (6), or they have been trimmed to 6-9 nt (7, 8). Wen and Gray (9) designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification (10). We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems (11, 12). The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10(-7) - 10(-8) M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

Catalytic DNAzymes: derivations and functions

Background: Although catalytic RNA enzymes (CRzs) are naturally occurring in many organisms, their DNA counterparts (CDzs) were developed by in vitro selection/evolution from random sequence libraries. Objective: To provide a brief overview of how CDzs have been selected in vitro, and of their properties and functions, as well as their possible future utility. Methods: We concentrated on examples of ‘direct’ selection of CDzs. Many CDzs have been used in biological settings, for example downregulation of target mRNAs, while many more recent applications use CDzs in biosensor and nanotechnology settings. Conclusions: Although much work has concentrated on using CDzs for regulating gene expression, their potential as nucleic acid medicines has diminished substantially, supplanted by simple antisense oligonucleotides and, more recently, by small interfering RNAs (siRNAs). It seems unlikely that CDzs will have clinical utility. In contrast, they are likely to have significant potential in the sensor/nanotechnology arena.