1. Gene therapy Fundamentals of Biotechnology

2. Principles and applications of therapy based on targeted inhibition of gene expression in vivo

3. Continue!! One way of treating certain human disorders is to selectively inhibit the expression of a predetermined gene in vivo. In principle, this general approach is particularly suited to treating cancers and infectious diseases, and some immunological disorders. In these cases, the basis of the therapy is to knock out the expression of a specific gene that allows disease cells to flourish, without interfering with normal cell function. For example, attention could be focused on selectively inhibiting the expression of a particular viral gene that is necessary for viral replication, or an inappropriately activated oncogene.oncogene

4. Continue! In addition to the above, targeted inhibition of gene expression may offer the possibility of treating certain dominantly inherited disorders. If a dominantly inherited disorder-----of a loss-of-function mutation, treatment may be difficult using conventional gene augmentation therapy. However, dominantly inherited disorders which arise because of a gain-of-function mutation may not be amenable to simple addition of normal genes. gain-of-function mutation Instead, it may be possible, in some cases, to inhibit specifically the expression of the mutant gene, while maintaining expression of the normal allele.allele

5. Continue! The expression of a selected gene might be inhibited by a variety of different strategies. One possible type of approach involves specific in vivo mutagenesis of that gene, altering it to a form that is no longer functional. Gene targeting by homologous recombination offers the possibility of site-specific mutagenesis to inactivate a gene. Gene targeting Instead, methods of blocking the expression of a gene without mutating it have been preferred.

6. Continue!! In principle, this can be accomplished at different levels: at the DNA level : (by blocking transcription);transcription at the RNA level: (by blocking post-transcriptional processing, mRNA transport or engagement of the mRNA with the ribosomes); or at the protein level: (by blocking post-translational processing, protein export or other steps that are crucial to the function of the protein).

8. Targeted inhibition of expression at the DNA level Under certain conditions, DNA can form triple-stranded structures, as occurs naturally in the case of a portion of the mitochondrial genome.genome The rationale of triple helix therapeutics is to design a gene-specific oligonucleotide that will have a high chance of base-pairing with a defined double-stranded DNA sequence of a specific target gene in order to inhibit transcription of that gene. transcription Binding of the single-stranded oligonucleotide to a pre-existing double helix occurs by Hoogsteen hydrogen bonds and certain bases are preferred. The most stable of such bonds are formed by a G binding to the G of a GC base pair and a T binding to the A of an AT base pair.

9. Targeted inhibition of expression at the RNA level. Antisense therapeutics involves binding of gene-specific oligonucleotides or polynucleotides to the RNA; in some cases, the binding agent may be a specifically engineered ribozyme, a catalytic RNA molecule that can cleave the RNA transcript. ribozyme

10. Targeted inhibition of expression at the protein level. Oligonucleotide aptamers and intracellular antibodies can be designed to specifically bind to and inactivate a selected polypeptide/protein.

11. Antisense oligonucleotides or polynucleotides can bind to a specific mRNA, inhibiting its translation and, in some cases, ensuring its destruction

12. Continue!! During transcription, only one of the two DNA strands in a DNA duplex, the template strand (or antisense strand), serves as a template for making a complementary RNA molecule.transcriptiontemplate strandantisense strand As a result, the base sequence of the single-stranded RNA transcript is identical to the other DNA strand (the sense strand), except that U replaces T.sense strand Any oligonucleotide or polynucleotide which is complementary in sequence to an mRNA sequence can therefore be considered to be an antisense sequence.

13. Continue!! Binding of an antisense sequence to the corresponding mRNA sequence would be expected to interfere with translation, and thereby inhibit polypeptide synthesis. Indeed, naturally occurring antisense RNA is known to provide a way of regulating the expression of genes in some plant and animal cells, as well as in some microbes.antisense RNA

14. Antisense oligodeoxynucleotides The use of artificial antisense oligodeoxynucleotides is often favored, simply because: they can be synthesized so simply. They can be transferred efficiently into the cytoplasm of cells using liposomes, and can migrate rapidly to the nucleus by passive diffusion through the pores of the nuclear envelope. Antisense oligodeoxyribonucleotides (ODNs) are preferred as they are generally less vulnerable to nuclease attack than oligoribonucleotides.

15. Continue!! Nevertheless, to protect against degradation by cellular exonucleases it is still usual to modify the oligonucleotides at their 3 ′ or 5 ′ ends e.g. by introducing more resistant phosphorothioate bonds where sulfur atoms are linked to phosphate groups instead of the normal oxygen atoms. Antisense ODNs are also preferred because they have the additional advantage of inducing the destruction of an mRNA to which they bind. This is so because an ODN-mRNA hybrid, like all DNA-RNA hybrids, is vulnerable to attack by a specific class of intracellular ribonuclease, RNase H which selectively cleaves the RNA strand.

16. Peptide nucleic acids Peptide nucleic acids (PNAs) are artificially constructed by attaching the bases found in nucleic acids to a pseudopeptide backbone. The normal phosphodiester backbone is entirely replaced with a polyamide (peptide) backbone composed of 2-aminoethyl glycine units. As a result, PNAs have improved flexibility compared to DNA or RNA, which permits more stable hybridization to DNA or RNA (by Watson-Crick hydrogen bonding). They are also more resistant to nuclease attack and may therefore be useful alternatives to conventional antisense oligonucleotides.

17. Ribozymes Some RNA molecules are able to lower the activation energy for specific biochemical reactions, and so effectively function as enzymes (ribozymes).ribozymes They contain two essential components: target recognition sequences (which base-pair with complementary sequences on target RNA molecules), and a catalytic component which cleaves the target RNA molecule while the base-pairing holds it in place. The cleavage leads to inactivation of the RNA, presumably because of subsequent recognition by intracellular nucleases of the two unnatural ends. Examples include human ribonuclease P and various ribozymes obtained from plant viroids (virus-like particles).

19. Intracellular antibodies (intrabodies) Antibody function is normally conducted extracellularly. Once synthesized, they are normally secreted into the extracellular fluid, or are transported to the surface of the B cell to act as an antigen receptor. Recently, however, it has been possible to design genes encoding intracellular antibodies, or intrabodies. Intrabodies can be directed to a particular cell compartment where they can bind to and inactivate a specific cell molecule such as a disease-causing protein, and so they have been envisaged to have potential for treating certain diseases, such as infectious diseases.

20. Oligonucleotide aptamers Oligonucleotide aptamers are oligonucleotides which can bind to a specific protein sequence of interest. Transfer of large amounts of a chemically stabilized aptamer into cells can result in specific binding to a predermined polypeptide, thereby blocking its function.

21. Mutant proteins Naturally occurring gain-of-function mutations can involve the production of a mutant polypeptide that binds to the wild-type protein, inhibiting its function. In many such cases, the wild-type polypeptides naturally associate to form multimers, and incorporation of a mutant protein inhibits this process.

22. Continue!! In some cases, gene therapy may be possible by designing genes to encode a mutant protein that can specifically bind to and inhibit a predetermined protein, such as a protein essential for the life-cycle of a pathogen. For example, one form of gene therapy for AIDS involves artificial production of a mutant HIV-1 protein in an attempt to inhibit multimerization of the viral core proteins.

23. Artificial correction of a pathogenic mutation in vivo is possible, in principle, but is very inefficient and not readily amenable to clinical applications

24. Continue!! Certain disorders are not easy targets for conventional gene therapy. For example, dominantly inherited disorders where a simple mutation results in a pathogenic gain of function cannot be treated by gene augmentation therapy, and targeted inhibition of gene expression may be difficult to achieve. An alternative to conventional gene therapy involves repair of a mutant sequence in vivo. In principle, this can be done by a variety of different experimental strategies at both the level of the mutant gene or its transcript.

25. Therapeutic repair at the DNA level One possible approach is to achieve correction of the genetic defect by therapeutic gene targeting.gene targeting Homologous recombination-based gene targeting low efficiency and there are formidable challenges in applying this technology to in vivo gene therapy. Other possibilities for therapeutic DNA repair utilize triple helix formation and peptide nucleic acids.

26. Therapeutic repair at the RNA level An alternative approach to gene targeting is to repair the genetic defect at the RNA level. One possibility is to use a therapeutic ribozyme.gene targetingribozyme One method envisages using a class of ribozyme known as group I introns,ribozyme which are distinguished by their ability to fold into a very specific shape, capable of both cutting and splicing RNA.splicing If a transcript has, for example, a nonsense or a missense mutation,missense mutation it may be possible to design specific ribozymes that can cut the RNA upstream of the mutation and then splice in a corrected transcript, a form of trans-splicing. upstreamsplicing

28. Continue!! Another possibility is therapeutic RNA editing.RNA editing This involves using a complementary RNA oligonucleotide to bind specifically to a mutant transcript at the sequence containing the pathogenic point mutation,point mutation and an RNA editing enzyme, such as double-stranded RNA adenosine deaminase, to direct the desired base modification.RNA editing

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