DNA Based Diagnosis and Treatment

1. PHM142 Fall 2012 Instructor: Dr. Jeffrey Henderson DNA Based Diagnosis and Treatment Name: Sign up Date: Elsa Chien, Wayne (Kin) Chun, Necole Chung and Silky Lee September 28, 2012

2. What is DNA Based Diagnosis? • detect presence of specific nucleic acid associated with a disease • high sensitivity and specificity, detects target DNA even at very low concentrations • tests don’t show pathogen viability • may not detect if pathogens and viruses mutate and change genetic structure

3. What is DNA Based Diagnosis? Cont’d . . . • false negatives may be caused by inaccurate selection of tissue sources • diagnose conditions such as cancer, cystic fibrosis and Down syndrome and infectious diseases such as anthrax and chlamydia Source: http://www.cartoonstock.com/newscartoons/cartoonists/aba/lowres/aban55l.jpg

4. Polymerase Chain Reaction • series of repetitive replication cycles • uses thermostable DNA polymerase to make several million copies of a targeted sequence • detects short deletions or insertions • Target amplification • DNA/RNA duplicated Source: http://www2.le.ac.uk/departments/emfpu/genetics/explained/pcr

5. Southern Blot Hybridization • DNA fragments separated by electrophoresis, then blotted on nylon filter • blotted nucleic acids fixed and hybridized using radioactively labelled ssDNA • used to diagnose genetic diseases from mutations • inheritance of a disease can also be predicted by polymorphic markers Source: http://www.molecularstation.com/dna/southern-blot/

6. Northern Blot Hybridization Source: http://www.molecularstation.com/rna/northern-blot/ • Uses mRNA as starting material • detects rearrangements within the coding region • Can be used to detect either large gene alterations or point mutations and polymorphisms altering a restriction site

7. Sanger/ Chain Termination Sequencing • Provides sequence of nucleotides on target DNA • Compares and identify microorganisms and mutations • Primer prepared with DNA polymerase and fluorescent dideoxynucleotides (ddNTP) Source: http://www.bio.davidson.edu/courses/bio111/seq.html

8. Shotgun Sequencing Source: http://www.bio.davidson.edu/COurses/genomics/method/hss2.gif • DNA sequence fragmented, cloned, sequenced then aligned • quick and inexpensive • can easily detect polymorphisms • may take many repeats to accurately align fragmented DNA • used to complete the Human Genome project

9. Gene Therapy • Use of DNA, to modify gene expression in cells for a therapeutic effect. • Two types of gene therapy • Somatic gene therapy • Germ line Gene therapy

10. Purpose of Gene Therapy • Provide a cure for various genetic diseases such as: • Cystic fibrosis • Haemophilia • Thalassemia • Provide an alternate mode of delivery of biological products to the body • Provide new therapy for disorders such as: • Cancer • Inflammatory diseases • Artherosclerosis

11. Germ Line Gene Therapy • gene therapy used to modify the inherited characteristics of germ cells. Therefore, descendants will also inherit this manipulation. • to this date, there has not been any clinical protocols involving gene transfer to germ line cells on humans due to ethical debate.

12. Somatic Gene Therapy • Gene therapy on somatic cells (biological cells other than reproductive cells i.e. germ cells) • Can be theoretically: • applied to any disease (as long as molecular pathogenesis is known) • designed to produce a therapeutic effect for a finite duration of action via pharmacokinetic properties • Therefore integration of new genetic material into the chromosomes does not have to be permanen

13. Gene Therapy – Viral Methods • Deliver genetic information to target cells by ex vivo or in vivo • Modified DNA injected into viral vectors • Ex vivo: target cells extracted from patients, therapeutic genes inserted and cells returned to patients • In vivo: viral vectors introduced to patients, targeting specific tissues and organs that require gene transfer

14. Adenoviruses • Non-enveloped icosahedral-shaped viruses that contain a linear dsDNA genome • Transfer transgenes to various cells, tissues and organs • Infect dividing and non-dividing cells • Large packaging capacity Source: http://www.daviddarling.info/encyclopedia/A/adenovirus_infection.html

15. Life Cycle of Adenoviruses • Virus binds to a coxsackievirus and adenovirus receptor (CAR) on host cell surface • Enters cell and releases virus genome into host nucleus but does not become integrated • Transcription of viral DNA begins and transient transgene expression occurs Source: Warnock, Daigre and Al-Rubeai, 2011.

16. Uses of Adenoviruses • Used for cancer treatment • In normal cells, DNA damage, cell cycle abnormalities or hypoxia can result in the development of tumours • Tumour suppressor genes are protective genes that limit the growth of tumours • Mutated or inactivated tumor suppressor genes can lead to cancer • In gene therapy, vectors are used to target cancer cells by transporting the tumour suppressor gene p53 • Overexpression of p53 inhibits tumour growth and induces apoptosis • Used to study stem cell differentiation, AIDS, cardiovascular diseases and pulmonary tuberculosis Source: http://en.wikipedia.org/wiki/File:P53_pathways.jpg

17. Adeno–associated virus (AAV) • Non-enveloped, icosahedral shaped • Linear ssDNA genome • AAV serotype 2 mostly used for gene therapy • Infect dividing and non-dividing cells Source: http://ksj.mit.edu/tracker/2009/05/ap-phil-inquirer-etc-new-tactic-immunity

18. Uses of Adeno-associated Viruses • Used in tissue engineering studies • Treat skin burns, excision and incision wounds • Vectors are stable in different tissues

19. Retroviruses • Capsid surrounded by lipid bilayer • contains surface and transmembraneglycoproteins encoded by retrovirus • Genome contains linear, ssRNA • 3 major genes: gag, pol and env, encode for proteins responsible for viral integration, replication and encapsulation • Only infect dividing cells Source: Warnock, Daigre and Al-Rubeai, 2011.

20. Life Cycle of Retroviruses • Bind to specific host cell receptors • Viral envelope fuses with host cell membrane, releases viral contents • Reverse transcription occurs, produces dsDNA and integrates into host cell genome (provirus) • Transcription occurs, provirus transcribed into mRNA, coded for viral proteins • Reassemble in cytoplasm and exit cell by budding Source: Ratcliffe, 2012. IMM250.

21. Uses of Retroviruses • Mostly in tissue repair and engineering • Construction of retroviral vectors require the removal of gag, pol and env genes, allowing the gene of interest to be inserted into the viral genome • Retroviral vectors have be used in attempt to treat X-linked severe combined immunodeficiency (X-SCID), but adverse effects have been reported in clincial trials

22. Gene Therapy Non-viral Methods • Low toxicity, lack of immune response and can be easily prepared • Not as efficient as viral methods due to: • - low and transient transgeneexpression • - limited ability to bind to surfaces of target cells • - ineffective in transporting the DNA through the nuclear membrane to the nucleus

23. Physical Approaches • Injection of naked plasmid DNA locally or systemically by a needle injection (ex. intramuscular) • An injection with physical or electrical forces applied - Create pores in the plasma membrane • Electroporation – transport DNA directly across the membrane with the application of an electric current Source: http://9e.devbio.com/article.php?ch=2&id=290 Source: http://spectrum.ieee.org/biomedical/devices/electroporation-knife-for-cancer

24. Chemical Approaches • Synthetic or natural compounds are used to form electrostatic interactions with plasmid DNA • Due to the negative nature of DNA, positively charged compounds such as cationic lipids and polymers form lipoplex and polyplex, respectively • Lipoplex and polyplex enter the cell by endocytosis and DNA release is based on different cellular conditions that affect the endosomes Source: http://mgl.scripps.edu/people/goodsell/mgs_art/mgs_art2/gaber2.html

25. Summary • DNA-based diagnostics use DNA analysis to detect nucleic acids associated with a disease • DNA Techniques for DNA diagnosis: • Polymerase Chain Reaction (PCR) • Hybridization • Sequencing • Gene therapy involves viral and non-viral methods • Somatic cell gene therapy • Germ line gene therapy • Modifies germ cells and thus descendants inherit this modification as well • Non-viral methods are not as effective as viral methods • Provides alternate mode of delivery of biological products to the body • Adenoviruses can be used as viral vectors to treat cancer • Adeno-associated viruses carry ssDNA and retroviruses carry ssRNA • both can be used as viral vectors for gene therapy

26. References • Bank, A. (1996). Human somatic cell gene therapy. BioEssays, 18(12), 999–1007. • Ferrari, M., Cremonesi, L., Carrera, P., & Bonini, P. A. (1996). Molecular diagnosis of genetic diseases. Clinical Biochemistry, 29(3), 201–208. doi:10.1016/0009-9120(95)02022-E • Grieger, J.C., & Samulski, R.J. (2012). Adeno-associated virus vectorology, manufacturing and clinical applications. Methods in Enzymology, 507, 229 – 254. • Ledley, F. D. (1994). Development in Somatic Gene Therapy, 3(9), 913–921. • Mountain, A. (2000). Gene therapy: the first decade. Trends in Biotechnology, 18(3), 119–128. • Limberis, M.P. (2012). Phoenix rising: gene therapy makes a comeback. Acta. Biochim. Biophys. Sin., 44(8), 632 – 640. • Matthews, J. Nuclear receptor I. PCL302, (October 31, 2011). • Matthews, J. Nuclear receptor III. PCL302, (November 08, 2011). • M. T. Tammi, The Principles of Shotgun Sequencing and Automates Fragment Assembly, Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm, Sweden, 2003. • Muldrew, K. (2009). Molecular diagnostics of infectious diseases. Current Opinion in Pediatrics, 21(1), 102-111. • Ratcliffe, M. Being a B cell: how antibodies neutralize pathogens. IMM250, (March 6, 2012). • Roth, J.A. (2006). Adenovirus p53 gene therapy. Expert Opin. Biol. Ther., 6(1), 55 – 61. • Salvi, M. (2011). Shaping Individuality: Human Inheritable Germ Line Gene Modification. Theoretical Medicine and Bioethics, 22(6), 527–542. • Sheridan, C. (2011). Gene therapy finds its niche. Nature Biotechnology, 29(2), 121 – 128. • Warnock, J.N., Daigre, C., & Al-Rubeai, M. (2011). Introduction to viral vectors. Methods in Molecular Biology, 737, 1 – 25. • Watcharanurak, K., Nishikawa, M., Takahashi, Y., & Takakura, Y. (2012). Controlling the kinetics of interferon transgene expression for improved gene therapy. Journal of Drug Targetting, 1 – 6. • Weber, J. L., & Myers, E. W. (1997). Human Whole-Genome Shotgun Sequencing. Genome Research, 7(5), 401–409.