Current Pharmaceutical Biotechnology, Vol. 5, No. 5, 2004
Contents
Genomics-Based
Anti-Cancer Drug Discovery
Guest
Editor: Gayathri R. Devi
Zebrafish
as a Genomics Research Model Pp.
409-413
Eleanor
Chen and Stephen C. Ekker
Anti-Genes:
siRNA, Ribozymes and Antisense Pp.
415-420
Kevin
J. Scanlon
Oligonucleotide
Mediated Gene Targeting in Mammalian Cells Pp. 421-430
Michael
M. Seidman
Neutrally
Charged Phosphorodiamidate Morpholino Antisense Oligomers: Uptake, Efficacy and
Pharmacokinetics Pp. 431-439
Vikram
Arora, Gayathri R. Devi and Patrick L. Iversen
The
Ins and Outs of RNAi in Mammalian Cells Pp. 441-450
M.
Banan and N. Puri
SV40
Pseudovirions as Highly Efficient Vectors for Gene Transfer and their Potential
Application in Cancer Therapy Pp. 451-458
Chava
Kimchi-Sarfaty and Michael M. Gottesman
Targeting
Steroid Hormone Receptor Pathways in the Treatment of Hormone Dependent Cancers Pp. 459-470
Y.J.
Ko and S.P. Balk
Role
of Genomics-Based Strategies in Overcoming Chemotherapeutic Resistance Pp. 471-480
M. Vijay Kumar, Robert Shirley, Yulin Ma and Ronald W. Lewis
Abstracts
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to top]
Zebrafish as a Genomics Research Model
Eleanor Chen and Stephen C. Ekker
The zebrafish (Danio rerio) is a recent addition to the genomic scientists’ repertoire of vertebrate animal model systems. Unlike simple invertebrates such as the fly or the nematode, this teleost maintains the biological and genomic complexity found in higher vertebrates. Furthermore, the zebrafish has many advantageous technical and genomic properties that open the door to experimental approaches not practical using more classical models. The zebrafish genome can be functionally accessed using both forward and reverse genetics based approaches. A notable recent addition to the zebrafish genomics toolbox is the development of morpholino-based antisense gene inhibition for sequence-based ‘knockdown’ screening. This method offers the opportunity to examine the role of significant subsets of the vertebrate genome for specific gene function in vivo. The zebrafish embryo can rapidly provide critical information for drug target discovery purposes when examined with an emphasis on clinically-relevant biological processes. Finally, the advent of chemical genetics in zebrafish suggests that, in addition to the identification and understanding of drug targets and their biology, this system will be a powerful tool in the direct development of novel pharmaceuticals in the near future.
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Anti-Genes: siRNA, Ribozymes and
Antisense
Kevin
J. Scanlon
Scientists have been working on strategies to selectively turn off specific genes in diseased tissues for the past thirty years. In the 1980’s, oligodeoxynucleotides (ODNs) with unique chemistries were tested with model systems both in vitro and in vivo with varying degrees of success. In the 1990’s, ribozymes with both antisense and catalytic properties were successfully introduced to the field. Ribozymes were shown to selectively knock down targeted genes in human tumors grown in mice but delivery issues for these therapeutic anti-genes limited their clinical utility. Short interfering RNA (siRNA) is currently the fastest growing sector of this anti-gene field for target validation and therapeutic applications. The siRNA field may have an opportunity to impact the clinic faster than antisense and ribozymes if the scientists can overcome the previous anti-gene limitations. Fortuitously, there have been a several developments involving the expansion of our genomic knowledge coupled with the rapid dissemination of disease genes by the digital revolution. This convergence of the knowledge of the human genome with the speed of digital communication will help facilitate swift changes in the detection and treatment of human illnesses. The anti-gene field is positioned to exploit this timely union of two distinct technologies. Anti-gene molecules have an opportunity to become a successful technology in understanding the human genome, as well as, enabling the development of efficacious gene therapy for human diseases in the near future. This review will characterize the advances in this field and address the challenges to the success of for the anti-gene technology.
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Oligonucleotide Mediated Gene Targeting
in Mammalian Cells
Michael M. Seidman
Gene targeting can be loosely defined as a process through which a specific chromosomal sequence is recognized and bound by a reagent designed for the purpose. The endpoint may be modulation of events at the target, such as transcription, or a permanent change in sequence at the site. A facile strategy for mammalian cells would have broad applications in basic and applied research, including gene therapy. Although approaches based on homologous recombination are routinely employed for transgenic animal construction, they are too laborious and inefficient for broader use. Consequently there has been a longstanding interest in developing effective synthetic reagents. Sequence recognition can be either at the level of a single strand or via the major or minor grooves, and specific approaches for each route are under development. In this review several oligonucleotide-based strategies will be discussed. These include single and double strand oligonucleotides designed to attack single strand targets, and triple helix forming olgonucleotides and peptide nucleic acids, intended for double stranded targets.
[Back to
top] Neutrally Charged Phosphorodiamidate Morpholino Antisense Oligomers:
Uptake, Efficacy and Pharmacokinetics
Vikram
Arora, Gayathri R. Devi and Patrick L. Iversen
Antisense technology constitutes development of sequence-specific DNA or RNA analogs that can block the activity of selected single-stranded genetic sequences and offer the potential of high specificity lacking in many current drug treatments. The sequencing of the human genome has greatly increased the potential of this approach. Antisense oligonucleotides, the most commonly used antisense approach, are unmodified or chemically modified single stranded RNA or DNA molecules specifically designed to hybridize to corresponding RNA by Watson-Crick binding. Phosphorodiamidate Morpholino oligomers (PMO) are a novel class of non-ionic antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation. PMOs have shown excellent efficiency and safety profile via various routes of administration in multiple animal and human studies. This review will summarize the preclinical studies with PMOs on the road to their development as therapeutic agents with particular emphasis on in vivo biodistribution and pharmacokinetics.
[Back to
top] The Ins and Outs of RNAi in Mammalian Cells
M. Banan and N. Puri
The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. RNAi in mammalian cells is achieved through introduction or expression of 21-23 bp small interfering RNAs (siRNAs) in cells or animals. Currently, there are six ways of producing siRNAs. siRNAs can be produced by chemical synthesis, in vitro transcription, or RNase III/Dicer digestion of long dsRNAs. Alternatively, they can be expressed in vivo from plasmids, PCR cassettes, or viral vectors that include a CMV or polymerase III (pol III) transcription unit. So far, these approaches have been used to create siRNAs for use in loss-of-function studies. However, it is clear that siRNAs also hold great promise as therapeutic tools. First, their activity seems to be very sequence-specific. Moreover, siRNAs could be modified in order to increase their stability and potency in vivo. Here, we will review the issues and findings related to siRNA design and production. Moreover, we will summarize new findings on siRNA specificity, modification, and delivery, which are critical to their use as therapeutic agents.
[Back to
top] SV40 Pseudovirions as Highly Efficient Vectors for Gene Transfer
and their Potential Application in Cancer Therapy
Chava Kimchi-Sarfaty and Michael M. Gottesman
Among viral and non-viral gene
delivery systems, SV40-based vectors show great promise in the cancer gene
therapy field. SV40 vectors very efficiently deliver genes such as anti-viral
agents, DNA vaccine, genes for chemoprotection (such as ABC transporters
genes), suicide genes and antiangiogenic genes. The recombinant SV40 vectors
can infect a wide variety of cells—dividing cells as well as non-cycling ones.
Most of the SV40-based vectors can incorporate larger transgenes than the
capacity of the SV40 wild-type, which is 5.2 kb; Moreover, in vitro
packaged vectors demonstrate efficient delivery of plasmids with a molecular
weight of up to 17.7 kb. SV40-based vectors carry some SV40 viral sequences,
but the SV40 in vitro-packaged vectors are free of any SV40 wild-type
viral DNA sequences. These vectors are prepared with nuclear extracts of SF9
insect cells containing the main viral capsid protein of the SV40 wild-type
virus, VP1. This review summarizes different strategies in which SV40 vectors
are used to deliver genes in vitro, to living mice, and to tumors
growing in nude mice.
[Back to
top] Targeting Steroid Hormone Receptor Pathways in the Treatment
of Hormone Dependent Cancers
Y.J.
Ko and S.P. Balk
Sex steroid hormones play a central role in the development and progression of prostate and breast cancers. The biological functions of these and other steroid hormones are mediated by a family of closely related steroid hormone receptors (SHRs), with the androgen receptor (AR) mediating the effects of testosterone and related androgens, and the classical estrogen receptor (ERa) mediating the effects of estradiol. Recent studies have begun to elucidate the complex pathways through which SHRs regulate gene expression, and their interaction with other cellular pathways. These studies have also begun to reveal molecular mechanisms underlying the diverse spectrum of effects mediated by steroid hormone analogues in different tissues. A major advance has been the finding that certain drugs induce unique conformational changes in SHRs that alter their interactions with transcriptional coactivator and corepressor proteins, resulting in cell type specific responses. These unique conformational changes appear responsible for the tissue specific effects of the selective estrogen receptor modulators (SERMs) in breast cancer. SHRs are clearly well established therapeutic targets in cancer, and drug development has continued to focus on agents that either block steroid hormone production or bind to and modulate their receptors. The identification of multiple proteins and pathways that mediate the downstream functions of SHRs may eventually provide additional therapeutic targets. This review outlines the basic biology of SHR structure and function, with a focus on AR and ERa. Hormonal therapies in prostate and breast cancer that directly target AR and ERa, respectively, are then presented and possible novel drug targets in the SHR pathway are discussed.
[Back to
top] Role of
Genomics-Based Strategies in Overcoming Chemotherapeutic Resistance
As cancer is being recognized as a failure of apoptosis, apoptosis-based strategies are being designed. Caspases are critical for the induction of apoptosis and their decreased expression is correlated with increased grade of cancer, while increased expression of caspases rendered the cancer cells susceptible to chemotherapy. However, the endogenous functions of caspases are inhibited by inhibitors of apoptosis (IAPs) that bind activated caspases. Methods to suppress the function of IAP induced apoptosis in chemo-resistant cancer cells. The function of IAPs is inhibited by Second Mitochondria-Derived Activator Of Caspase (Smac) or Direct IAP Binding Protein With Low Pi (DIABLO). Upon apoptotic stimulus Smac/DIABLO is released from the mitochondria, which binds to IAPs and inhibits their caspase-binding activity. Overexpression of Smac/DIABLO sensitized neuroblastoma to TRAIL (TNFa-Related Apoptosis-Inducing Ligand). Activation of TRAIL pathway has become an important method of inducing apoptosis except in TRAIL-resistant cells. However, treatment of these cells with other cytotoxic drugs sensitizes them to TRAIL, providing effective therapeutic advantages. In addition to activating apoptotic pathways, inhibiting or suppression of cell proliferation is necessary to sensitize cancer cells to apoptosis. Critical among these proteins are NFkB and Akt. NFkB blocked apoptosis by interfering with the function of TNFa/TRAIL and/or through the activation of antiapoptotic proteins of the Bcl2 family. Similarly, Akt mediate cell survival via the regulation of cell survival proteins and by blocking the function of proapoptotic Bad by phosphorylation. Altering the expression of Akt by dominant negative constructs or by expression of PTEN interferes with Akt function. In summary, this review points out the complexity of interactions of the cell survival and death pathways and highlights some methods to manipulate them to achieve therapeutic advantage.