Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Molecular Biotechnology of Microbiological Systems
Gene Therapy
Oligonucleotide-Based Therapeutics

Most ex vivo and In Vivo Gene therapy protocols employ cloned genetic constructs that restore the functional form of a protein that is either not synthesized in the patient's body or is produced in a defective form. However, many human diseases—such as Cancer, inflammatory conditions, viral infections, and parasitic diseases—are associated, conversely, with the overexpression of a normal protein. To treat such conditions, therapeutic systems utilizing oligonucleotides have been developed. A short oligonucleotide can hybridize with a specific gene or mRNA, thereby downregulating METABOLISM/31.html">Transcription or Translation and reducing The amount of the synthesized protein responsible for the pathology. An oligonucleotide that hybridizes with the gene itself and blocks its transcription is termed an "antigene" oligonucleotide, whereas one that hybridizes with the corresponding mRNA is called "antisense." The transcription and translation of a target gene can also be suppressed by an oligonucleotide that binds to a transcription factor controlling the expression of that specific gene. Double-stranded oligonucleotides that attach to DNA-binding Proteins can likewise be used to prevent the transcriptional activation of specific genes. Furthermore, synthetic DNA molecules can be engineered to bind to specific target proteins that are not naturally DNA-binding, thereby blocking their normal function. Finally, to decrease the levels of a specific mRNA and the protein translated from it, one can modify ribozymes—natural RNA sequences that bind to specific RNA molecules and cleave them. In the future, nucleic acid-based Pharmaceuticals will likely find widespread application, with antisense sequences—and particularly antisense oligonucleotides—serving as the primary focus of scientific research and clinical trials.

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Fig. 21.12. Combined approach using GCV-HSVtk therapy and gene immunotherapy. Tumor Cells were transduced in vivo with the Herpes simplex virus thymidine kinase (HSVtk) gene and a cytokine gene. Transduced tumor cells synthesizing the cytokine (Cyt) recruit immune cells, whereas those synthesizing thymidine kinase (TK) phosphorylate ganciclovir. Phosphorylated ganciclovir passes through Intercellular junctions (black rectangles) into neighboring cells and destroys them by binding to their DNA (X).

Synthesis of "antisense" mRNA in vivo

"Antisense" RNA intended for use as a therapeutic agent must bind to a specific mRNA and inhibit the translation of its encoded protein, thereby suppressing the pathological process (Fig. 21.13). Expression vectors carrying DNA inserts oriented such that their transcripts are antisense to the target mRNA have been used to produce such RNAs. Examples include episomal expression vectors harboring cDNAs for Insulin-like growth factor 1 (IGF-1) or its receptor (IGF-IR) in reverse orientation under the control of a metallothionein promoter activated by ZnSO₄. IGF-1 is overexpressed by malignant glioma cells, the most common type of Brain tumor, whereas IGF-1R is overexpressed by prostate carcinoma cells.

Fig. 21.13. Inhibition of specific mRNA Translation using antisense Nucleic Acids. A. The cDNA is inserted into an expression vector in reverse orientation, and the resulting genetic construct is introduced into cells, where antisense RNA Synthesis takes place. This RNA hybridizes with the target mRNA and blocks translation. B. An antisense oligonucleotide is introduced into The Cell, hybridizing with the target mRNA and blocking translation. Abbreviations: p, promoter; pa, polyadenylation signal; A, intron; 1 and 2, exons.

A vector directing the synthesis of FGF-1 antisense mRNA was transfected into a glioma cell culture. In the absence of ZnSO₄ in the culture medium, the tumor's overexpression of IGF-1 persisted, whereas The addition of ZnSO₄ to the medium abolished it. In another experiment, the researchers studied the effects of injecting rats with untransfected glioma cells versus cells transfected with IGF-1 antisense cDNA. Tumors developed in the first case, but not In the second.

When rat prostate carcinoma cells transfected with IGF-1R antisense cDNA were injected into mice, only small tumors formed or none at all. Conversely, injection of untransfected cells or cells transfected with normal IGF-1R cDNA resulted in the growth of large tumors. Presumably, in both instances, the antisense RNA hybridizes with the complementary mRNA and inhibits the translation of IGF-1 and IGF-1R, thereby preventing tumor cell proliferation.

"Antisense" oligonucleotides as therapeutic agents

The therapeutic efficacy of synthetic antisense oligonucleotides depends on the Specificity of their Hybridization with an accessible site on the target mRNA, their resistance to cellular Nucleases, and the availability of an efficient intracellular delivery system. Sequences of 15–20 NUCLEOTIDES hybridize with unique mRNAs with fairly high specificity. Potential target sites are identified by screening a panel of antisense oligonucleotides using a cell culture that synthesizes the target mRNA. This involves electrophoretic Separation of cellular proteins radiolabeled during translation, followed by

autoradiography to determine in the presence of which antisense oligonucleotide the synthesis of a specific protein is diminished. There are no universal criteria for selecting the best target sites across different RNA transcripts. Oligonucleotides complementary to the 5' or 3' ends of mRNA, exon-intron junctions, or even double-stranded regions can prove effective. Because oligodeoxynucleotides are degraded by intracellular nucleases, it is crucial to protect them against nuclease activity without compromising their ability to hybridize with the target. This can be achieved by specific modifications of the pyrimidine bases and the deoxyribose sugar (Fig. 21.14). For instance, in the most widely used antisense oligonucleotides today, a non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur group (Fig. 21.14, B), yielding a phosphorothioate linkage. Oligonucleotides modified in this manner are Water-soluble, negatively charged, and resistant to endonuclease Cleavage. Upon hybridization with the target site, they form RNA–DNA duplexes that activate Ribonuclease H (RNase H), an endogenous enzyme that cleaves the mRNA within the hybrid molecule. The first clinical trials of such first-generation oligonucleotide drugs have already been conducted. Their targets include the RNAs of cytomegalovirus and HUMAN IMMUNODEFICIENCY VIRUS, as well as mRNAs of genes responsible for cancer, intestinal disorders, and other diseases.

Antisense oligonucleotides with phosphoramidite and polyamide (peptide) linkages have also been synthesized (Fig. 21.14, C and D). Such molecules exhibit high resistance to nuclease degradation. Chemical groups attached to the 2'-carbon atom of the sugar moiety and the C-5 position of Pyrimidines likewise protect antisense oligonucleotides and facilitate their binding to the target site (Fig. 21.14, E and F). The full advantages of these and other modifications are currently under intensive investigation.

The cellular uptake of antisense oligonucleotides can be substantially enhanced by encapsulating them in Liposomes. Such a highly efficient delivery system permits The Use of antisense oligonucleotides at low concentrations. Furthermore, conjugating liposomes with cell-specific binding sites makes targeted delivery of the oligonucleotides feasible.

Fig. 21.14. Modifications of antisense oligonucleotides. A. Phosphodiester linkage. B. Phosphorothioate linkage. C. Phosphoramidite linkage. D. Polyamide linkage (peptide nucleic acid). E. 2'-O-methylribose. F. C-5-propynylcytosine.

Preclinical studies have demonstrated that antisense oligonucleotides are highly potent therapeutic agents. Their potential application in treating coronary and carotid artery restenosis—which leads to myocardial infarctions and strokes—has been investigated. Although angioplasty (balloon catheter dilation of Arteries) is frequently used in such cases, restenosis recurs in approximately 40% of patients within six months because angioplasty stimulates smooth Muscle cell proliferation and Extracellular matrix secretion into the tunica intima at the dilation site. In one experiment, phosphorothioate antisense oligonucleotides complementary to mRNAs encoding proteins essential for the mammalian Cell Cycle were administered to rat carotid arteries following angioplasty; As a result, The rate of restenosis was reduced by 90%. Smooth muscle cell proliferation also occurs in atherosclerosis, Diabetes Mellitus, and complications following coronary artery bypass grafting. It is likely that all these conditions can be managed using similar approaches.

Antisense oligonucleotides can likewise be employed to treat VIRAL INFECTIONS AND malaria. Moreover, the results of Phase I clinical trials for Crohn's disease Treatment via oral administration of an antisense oligonucleotide demonstrated a pronounced therapeutic effect with no noticeable adverse side effects. In this case, the target mRNA encoded intercellular adhesion molecule 1, which is overproduced in patients with Crohn's disease. The efficacy of this same oligonucleotide is planned to be evaluated for treating other inflammatory conditions, such as rheumatoid Arthritis, psoriasis, and Ulcerative Colitis.

In theory, antisense oligonucleotides can form a triple helix with chromosomal DNA targets and thereby block transcription. However, the specificity of antigene oligonucleotides does not yet meet the rigorous standards established for pharmaceutical drugs.

Protein-binding oligonucleotides: the Thrombin aptamer

The expression of a target gene can be blocked not only through antisense therapy but also by introducing into the cell an oligonucleotide that binds to a transcription or translation factor, although this approach remains insufficiently explored. Furthermore, because nucleic acids are capable of binding to proteins, one can synthesize an oligonucleotide (known as an aptamer) designed to attach to a specific protein—one that is not normally associated with any nucleic acids—and block its function. For instance, the thrombin aptamer holds promise as an affordable prophylactic agent against thrombus formation during various surgical Procedures.

To generate this aptamer, the researchers used a combinatorial library of chemically synthesized oligonucleotides consisting of 18-nucleotide flanking regions (primers) and a central 60-nucleotide segment where any of the four nucleotides could occur at each of the 60 positions. Theoretically, such a library contains approximately 1.3 × 10³⁶ (4⁶⁰) oligonucleotides with distinct central sequences. The sample was passed through a Column containing immobilized thrombin molecules; the oligonucleotides that bound to thrombin were eluted and reapplied to the thrombin-conjugated column. This Procedure was repeated at least three times. The final pool of thrombin aptamers was amplified by PCR and cloned, after which the physical and biological Properties of Individual clones were determined. Aptamers exhibiting high affinity, specificity, and antithrombin activity were selected for more detailed analysis.

This approach yielded an effective thrombin aptamer. Unfortunately, due to its short in vivo half-life, it can only be used for transient inhibition of thrombin function (such as during cardiopulmonary bypass) and is unsuitable for Applications requiring prolonged anticoagulation (e.g., angioplasty). Obviously, the aptamer identification procedure described here can be adapted for other target proteins as well.

Ribozymes as therapeutic agents

Ribozymes are natural RNA molecules possessing catalytic activity (RNA Enzymes); their substrate-binding domain attaches to a complementary target RNA via Hydrogen Bonds and possibly other interactions, while their catalytic domain cleaves the target at a specific site. By modifying the substrate-binding sequence, one can generate a ribozyme specific for a given mRNA (Fig. 21.15). Designing a therapeutic ribozyme is a challenging process, primarily due to the difficulty of producing large quantities of synthetic RNA and maintaining it in its native conformation within the target cell. In one experiment, an oligodeoxynucleotide was synthesized containing a catalytic domain (approximately 20 nucleotides) flanked by sequences capable of hybridizing with the target mRNA (which also served as primers); this construct was amplified, inserted into a eukaryotic expression vector, and transfected into cells. The ribozyme produced via transcription cleaved the target mRNA and suppressed the Introduction/27.html">Translation of the protein responsible for a given disease. Engineered ribozymes hold potential for the treatment of cancer and viral infections.

Fig. 21.15. Cleavage of mRNA by a ribozyme. The ribozyme, whose substrate-binding domain has been genetically engineered, hybridizes with the target mRNA and cleaves it at a specific site (indicated by the arrow). (Adapted from Tone et al., In Vivo 7: 471–476, 1993.)

Natural DNA enzymes (deoxyribozymes) have not yet been discovered, but oligodeoxynucleotides with catalytic activity have already been synthesized. The advantage of deoxyribozymes is that their production does not require an expression vector: DNA Enzymes can be packaged into liposomes and delivered into the target cell. However, The Development of efficient DNA enzymes is still in its early stages.