Contents
Novel
Therapeutic Perspectives: The Targeted Inhibition of Genes and Proteins
Executive Editor: Felix Hoppe-Seyler
Oligonucleotide-Directed Mutagenesis and
Targeted Gene Correction: A Mechanistic Point of View Pp.445-463
Antisense Strategies Pp.465-487
Stanley
T. Crooke
Ribozymes in the Age of Molecular
Therapeutics Pp.489-506
Sepideh
Bagheri and Mohammed Kashani-Sabet
Silencing of Disease-related Genes by Small
Interfering RNAs Pp.507-517
U.
Fuchs, C. Damm-Welk and A. Borkhardt
Intracellular Antibodies as Specific Reagents
for Functional Ablation: Future Therapeutic Molecules Pp.519-528
M.
N. Lobato and T. H. Rabbitts
Peptide Aptamers: Specific Inhibitors of
Protein Function Pp.529-538
Felix
Hoppe-Seyler, Irena Crnkovic-Mertens, Evangelia Tomai and Karin Butz
Therapeutic Antibodies Pp.539-547
Bernd
Groner, Cord Hartmann and Winfried Wels
Abstracts
[Back to top] Oligonucleotide-Directed Mutagenesis and
Targeted Gene Correction: A Mechanistic Point of View
Within the last
decade, a number of nucleic acid-based gene targeting strategies have been
developed with the ultimate goal to cure human genetic disorders caused by
mutations. Thus far, site-directed gene targeting is the only procedure that
can make predefined alterations in the genome. The advantage of this approach
is that expression of the corrected gene is regulated in the same way as a
normal gene. In addition, targeted specific mutations can be made in the genome
for functional analysis of proteins. Several approaches, including chimeric
RNA-DNA oligonucleotides, short single-stranded oligonucleotides, small
fragment homologous replacements, and triple-helix-forming oligonucleotides
have been used for targeted modification of the genome. Due to the absence of
standardized assays and mechanistic studies in the early developmental stages
of oligonucleotide-directed gene alteration, it has been difficult to explain
the large variations and discrepancies reported. Here, we evaluate the progress
in the field, summarize the achievements in understanding the molecular
mechanism, and outline the perspective for the future development. This review
will emphasize the importance of reliable, sensitive and standardized assays to
measure frequencies of gene repair and the use of these assays in mechanistic
studies. Such studies have become critical for understanding the gene repair
process and setting realistic expectations on the capability of this
technology. The conventionally accepted but unproven dogmas of the mechanism of
gene repair are challenged and alternative points of view are presented.
Another important focus of this review is the development of general selection
procedures that are required for practical application of this technology.
[Back to top] Antisense Strategies
Stanley
T. Crooke
Antisense
technology exploits oligonucleotide analogs to bind to target RNAs via Watson-Crick
by hybridization. Once bound, the antisense agent either disables or induces
the degradation of the target RNA. Antisense agents may also be used to alter
splicing.
Developing
antisense technology involves the creation of a new pharmacology. The
receptors, pre- and m- RNAs, had never been studied before as sites for drug
binding and action. The drugs, oligonucleotide analogs, had never made or
tested as drugs before and no medicinal chemistry had been performed. The
receptor binding mechanism, Watson-Crick hybridization had never been
demonstrated as feasible to exploit from a pharmacological perspective. The
post-receptor binding events were literally unknown and unexplored.
During the past
decade or more, substantial progress has been made in developing antisense
pharmacology. A great deal has been learned about the basic mechanisms of
antisense, the medicinal chemistry, the pharmacological, pharmacokinetic and
toxicological properties of antisense molecules. Antisense technology has
proven of great value in gene functionalization and target validation. With one
drug marketed, VitraveneŽ, and approximately 20 antisense drugs in clinical
development, it appears that antisense drugs may prove of value in the
treatment of a wide range of diseases.
In this review,
the progress is summarized, the limitations of the technology discussed and the
future considered.
[Back to top] Ribozymes in the Age of Molecular
Therapeutics
Sepideh
Bagheri and Mohammed Kashani-Sabet
Ribozymes are RNA
molecules capable of sequence-specific cleavage of other RNA molecules. Since
the discovery of the first group I intron ribozyme in 1982, new classes of
ribozymes, each with their own unique reaction, target site specifications, and
potential applications, have been identified. These include hammerhead,
hairpin, hepatitis delta, varkud satellite, groups I and II intron, and RNase P
ribozymes, as well as the ribosome and spliceosome. Meanwhile, ribozyme
engineering has enabled the in vitro selection of synthetic ribozymes
with unique properties. This, along with advances in ribozyme delivery methods
and expression systems, has led to an explosion in the potential therapeutic
applications of ribozymes, whether for anti-cancer or anti-viral therapy, or
for gene repair.
[Back to top] Silencing of Disease-related Genes by Small
Interfering RNAs
U.
Fuchs, C. Damm-Welk and A. Borkhardt
In recent years a
new mechanism of posttranscriptional gene silencing has been discovered and
named RNA interference. The interference is based on mRNA degradation mediated
by small double-stranded RNA molecules approximately 21 nucleotides in length,
the so-called short interfering or siRNAs. These molecules are produced from
long dsRNAs by Dicer, a dsRNA-specific endonuclease, and cause specific
degradation of their mRNA-targets by Watson-Crick base-pairing within a 300 kD
multi-enzyme complex named RISC. RNAi is highly conserved between plants and
animals of various phyla including mammals. The high sequence-specificity of
RNAi makes it a new, promising tool in gene-function analysis as well as in
potential therapeutics. In this review the discovery and molecular background
of RNAi are summarized and possible fields of application pointed out.
[Back to top] Intracellular Antibodies as Specific Reagents
for Functional Ablation: Future Therapeutic Molecules
M. N. Lobato and T. H. Rabbitts
The use of
antibodies in medicine and research depends on their specificity and affinity
in the recogniton and binding of individual molecules. However, these
applications are limited to the extracellular targets. Advances in antibody
engineering has allowed the manipulation of the antibody segments containing
the antigen-binding regions and generation of small fragments that can be
stably expressed in cells. These entities are called intracellular antibodies
or intrabodies and have being successfully applied, mainly in the scFv format,
to inhibit the function of intracellular target proteins in specific cellular
compartments. As new techniques to select and isolate intrabody fragments have
been developed, intrabodies are beginning to be used to interfere with the
function of a greater number of relevant disease targets. Just as monoclonal
antibodies are opening a new era in human therapeutics, intrabodies promise a
new prospective for antibody tools for therapy and research. Their varied mode
of action gives intrabodies great potential in different approaches in the treatment
of human diseases, as well as in the area of functional genomics for
characterisation of novel gene products and subsequent validation as potential
drug targets. While techniques for identifying functional intrabodies have
improved, there are still many significant problems to be overcome before
intrabodies can actually be used in treatment of diseases such as cancer, AIDS
or neuro-degenerative disorders.
[Back to top] Peptide Aptamers: Specific Inhibitors of
Protein Function
Felix
Hoppe-Seyler, Irena Crnkovic-Mertens, Evangelia Tomai and Karin Butz
In recent years,
peptide aptamers have emerged as novel molecular tools that are useful for both
basic and applied aspects of molecular medicine. Due to their ability to specifically
bind to and inactivate a given target protein at the intracellular level, they
provide a new experimental strategy for functional protein analyses, both in
vitro and in vivo. In addition, by using peptide aptamers as
“pertubagens”, they can be employed for genetic analyses, in order to identify
biochemical pathways, and their components, that are associated with the
induction of distinct cellular phenotypes. Furthermore, peptide aptamers may be
developed into diagnostic tools for the detection of a given target protein or
for the generation of high-throughput protein arrays. Finally, the peptide
aptamer technology has direct therapeutic implications. Peptide aptamers can be
used in order to validate therapeutic targets at the intracellular level. Moreover,
the peptide aptamer molecules themselves should possess therapeutic potential,
both as lead structures for drug design and as a basis for the development of
protein drugs.
[Back to top] Therapeutic Antibodies
Bernd
Groner, Cord Hartmann and Winfried Wels
Monoclonal
antibodies had the lure of drugs very much since their first description. The
ability to bind to a predetermined chemical structure stimulated the
imagination of drug discoverers and developers. Nevertheless it took many years
before a drug was registered which started to make good on the promise. The
complexity of the molecule, made up of four polypeptide chains, its large
molecular weight, its multiple and versatile functional domains and its mouse
origin initially were obstacles for the production and the utilisation. Also
the selection of appropriate target structures on the surface of cells turned
out be difficult. Many of these difficulties have been overcome. The
replacement of most of the murine sequences with equivalent human sequences and
the concomittant decrease in immunogenicity, and the identification of cell
surface components which are causative and limiting in cellular transformation
have made monoclonal antibodies valuable weapons in the fight against cancer.
Multiple mechanisms of monoclonal antibody action are being exploited for this
purpose. Antibodies can sequester growth factors and prevent the activation of
crucial growth factor receptors. A monoclonal antibody directed against the vascular
endothelial growth factor (VEGF) has been shown to be a potent
neo-vascularisation inhibitor (bevacizumab). An antibody against the
extracellular domain of the EGF receptor prevents the binding of the ligand to
the receptor and thereby its activation (cetuximab). EGFR activity, however, is
absolutely required for the survival and proliferation of certain human tumour
cells. An antibody which interferes with the dimerisation of the ErbB2 and the
ErbB3 members of the EGF receptor family prevents the association of a most
potent signaling module (pertuxumab). The signals emenating from this dimer
determine many phenotypic properties of e.g. human breast cancer cells. A
monoclonal antibody also directed against ErbB2 has been most successful,
clinically and commercially (trastuzumab). This antibody interferes with
signals generated by the receptor and causes the arrest of the cell cycle in
tumour cells. In addition, it recruits immune effector cells as cytotoxic
agents. Finally, monoclonal antibody derivatives, single chain Fv fragments,
have been used as a basis for the construction of recombinant tumour toxins.
These molecules harness the exquisite binding specificity of the antibodies and
combine them with the toxic principles of bacteria.