‫Lecture 10 .pptxfyhjjhghkkbggjkoojbhfds

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1. Genetic Engineering Continue

2. Assist. Prof. Dr. Ali Al- Yafai Assistant Professor of Diagnostic & Molecular Microbiology

3. Gene therapy

4. Gene therapy Genetic diseases seriously threaten human health and have always been one of the refractory conditions facing humanity. Theoretically, gene therapy is the permanent solution for genetic diseases. Gene therapy is the process of inserting genes into cells to treat diseases. Gene therapy is the process of replacing the defective gene with exogenous DNA and editing the mutated gene at its native location.

5. Gene therapy The introduction of a functional gene into the patient’s cells will encode proteins and correct the deficiencies that occur in genetic diseases . A large number of genetic disorders and other diseases are at various stages of gene therapy trials e.g. sickle-cell anemia, cystic fibrosis, AIDS, cancer. In case of gene inhibition therapy, the antisense gene inhibits the expression of the dominant gene. The major challenges of gene therapy are immunogenicity, mutagenicity, and lack of sustainable therapeutic benefit.

6. Gene therapy There are two approaches to achieve gene therapy: Somatic cell gene therapy Germ cell gene therapy

7. Gene therapy Somatic cell gene therapy The nonreproductive (non-sex ) cells of an organism are referred to as somatic cells. These are the cells of an organism other than sperm or egg cells, e.g ., bone marrow cells, blood cells, skin cells, intestinal cells. In essence, somatic cell gene therapy involves the insertion of a fully functional and expressible gene into a target somatic cell to correct a genetic disease permanently .

8. Gene therapy Germ cell gene therapy The reproductive (sex ) cells of an organism constitute germ cell line. Gene therapy involving the introduction of DNA into germ cells is passed on to the successive generations For safety, ethical and technical reasons, germ cell gene therapy is not being attempted at present.

9. Gene therapy There are two types of gene therapies. Ex vivo gene therapy In vivo gene therapy Ex vivo gene therapy Ex vivo gene therapy involves the transfer of genes in cultured cells which are then reintroduced into the patient.

10. Gene therapy Ex vivo gene therapy The ex vivo gene therapy can be applied to only selected tissues (e.g ., bone marrow) whose cells can be cultured in the laboratory. The procedure basically involves the use of the patient’s own cells for culture and genetic correction , and then their return back to the patient .

11. Gene therapy Ex vivo gene therapy The technique of ex vivo gene therapy involves the following steps Isolate cells with genetic defect from a patient. Grow the cells in culture. Introduce the therapeutic gene to correct gene defect. Select the genetically corrected cells (stable transformants ) and grow. Transplant the modified cells to the patient .

12. Gene therapy Ex vivo gene therapy The carrier particles or molecules used to deliver genes to somatic cells are referred to as vectors . The important vectors employed in ex vivo gene therapy are Viruses Human artificial chromosome Bone marrow cells.

13. Gene therapy Ex vivo gene therapy A number of viruses have been used in human gene therapy, including retrovirus, lentivirus , adenovirus, adeno -associated virus, herpes simplex virus, cytomegalovirus, and vaccinia virus In retrovirus as example: its genetic material is RNA . As the retrovirus enters the host cell, it synthesizes DNA from RNA (by reverse transcription). The so formed viral DNA (referred to as provirus) gets incorporated into the DNA of the host cell. The proviruses are normally harmless.

14. Gene therapy Ex vivo gene therapy However, there is a tremendous risk, since some of the retroviruses can convert normal cells into cancerous ones. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. The HAC is a synthetic chromosome which can carry large DNA (one or more genes) and can replicate with other chromosomes.

15. Gene therapy Ex vivo gene therapy Bone marrow contains totipotent embryonic stem (ES) cells. These cells are capable of dividing and differentiating into various cell types (e.g., red blood cells, platelets, macrophages, osteoclasts, B- and T-lymphocytes). For this reason, bone marrow transplantation is the most widely used technique for several genetic diseases.

16. Gene therapy In vivo gene therapy The direct delivery of genes into the cells of a particular tissue is referred to as in vivo gene therapy. Many tissues are the potential candidates for this approach. These include liver, muscle , skin, spleen, lung, brain and blood cells. Gene delivery can be carried out by viral or nonviral vector systems.

17. Gene therapy In vivo gene therapy The success of in vivo gene therapy mostly depends on the following parameters The efficiency of the uptake of the remedial (therapeutic ) gene by the target cells. Intracellular degradation of the gene and its uptake by nucleus. The expression capability of the gene.

18. Gene therapy Currently, gene therapy drugs such as small Interfering RNA ( siRNA ), antisense oligonucleotide, CRISPR/Cas9 system, plasmid DNA and MicroRNA ( miRNA ) have shown great potential in biomedical applications. In general, gene therapy is carried out by introducing a therapeutic gene to produce the defective or the lacking protein. But there are certain disorders (cancer, viral and parasitic infections , inflammatory diseases) which result in an overproduction of certain normal proteins.

19. Gene therapy It is possible to treat these diseases by blocking transcription using a single-stranded nucleotide sequence ( antigene oligonucleotide) that hybridizes with the specific gene, and this is called antigene therapy. Antisense therapy refers to the inhibition of translation by using a single stranded nucleotide (antisense oligonucleotide).

20. Gene therapy it is also possible to inhibit both transcription and translation by blocking ( with oligonucleotides ) the transcription factor responsible for the specific gene expression. siRNA molecules interfere with the expression of genes by binding to messenger RNA (mRNA) after it's been transcribed from the genome. The antiviral drugs such as ganciclovir , Acyclovir and zidovudine are Nucleosideanalogue that based on their ability to inhibit DNA synthesis by enzymes.

21. Transgenic organisms

22. Transgenic organisms The transgenesis technique involves the introduction of foreign DNA sequences into the genome of transfected cells and ensuring that the DNA sequences are integrated and transmitted to the offspring. A transgenic animal is one whose genome has been changed to carry genes from another species or to use techniques for animal genome editing for specific traits. Transgenic refers to an organism or cell whose genome has been altered by the introduction of one or more foreign DNA sequences from another species by artificial means.

23. Transgenic organisms Transgenic organisms are genetically engineered to carry transgenes—genes from a different species—as part of their genome. Transgenic animals: Transgenic means containing genetic material into which DNA from a different organism has been artificially introduced. A mouse was the first successful transgenic animal, then pigs, sheep, cattle, and rabbits came a few years later.

24. Transgenic organisms The foreign-interested genes that will be used in animal transgenic techniques are prepared using a variety of methods. The produced gene of interest is placed into a variety of vectors, including yeast artificial chromosomes, bacterial plasmids, and cosmids . Several techniques, including heat shock, electroporation, viruses, the gene gun, microinjection, and liposomes, are used to deliver the created vector, which includes the interesting gene, into the host cell.

25. Transgenic organisms Transgenic mice, rabbits, pigs, and sheep have been created by incubating sperm cells with foreign DNA and fertilizing them in vitro or in vivo.

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