Gene Therapy - Problems and Challenges

1. Gene Therapy - Problems and Challenges Ömer Faruk Bayrak

2. Gene Therapy Background to Gene Therapy Potential Benefits Perceived Hazards and Risks Regulations Future? 2

3. What’s gene therapy? • Imagine that you accidentally broke one of your neighbor's windows. Many medical conditions result from flaws, or mutations, in one or more of a person'sgenes.So, if a flawed gene caused our "broken window," can you "fix" it? What are your options? • Stay silent: ignore the genetic disorder and nothing gets fixed. • Try to treat the disorder with drugs or other approaches: depending on the disorder, treatment may or may not be a good long-term solution. • Put in a normal, functioning copy of the gene: if you can do this, it may solve the problem! • Stay silent: no one will ever find out that you are guilty, but the window doesn't get fixed. • Repair it with some tape: not the best long-term solution. • Put in a new window: not only do you solve the problem, but also you do the honorable thing.

4. Background • In the 1980s, advances in molecular biology had already enabled human genes to be sequenced and cloned. Scientists looking for a method of easily producing proteins, such as the protein deficient in diabetics — insulin, investigated introducing human genes to bacterial DNA. The modified bacteria then produce the corresponding protein, which can be harvested and injected in people who cannot produce it naturally. • Scientists took the logical step of trying to introduce genes straight into human cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia, optic nerve disease1, wound repair and regeneration2, and cardiovascular disease3. • However, this has been much harder than modifying simple bacteria, primarily because of the problems involved in carrying large sections of DNA and delivering it to the right site on the genome.

5. Gene Therapy Definition The deliberate introduction of genetic material into human somatic cells for therapeutic, prophylactic or diagnostic purposes Addition of EXTRA genes Aim is to cure disease (or at least help the patient) First introduction of gene-modified cells into a patient was in 1989 First gene therapy product approved for market in 2004 Still very experimental and early in its development 5

6. What is gene therapy? Why is it used? • Gene therapy = Introduction of normal genes into cells that contain defective genes to reconstitute a missing protein product • GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized  to improve a genetic disorder

7. Diseases for applying gene therapy Disease Defect Target cell Severe combined Bone marrow cells or immunodeficiency T-lymphocytes Hemophilia Liver, muscle Cystic fibrosis Lung Cells Cancer Many cell types Neurological diseases Parkinson’s/ Alzheimers Nerve Cells Infectious diseases AIDS, hepatitis B White Blood Cells

8. Defining Mutation Type Oncogenes (active) • Point mutations • Amplification (Duplication) • Formation of chimeric gene TumorSupressor Gene (inactivate) • Lossof heterozygosity

9. Gene therapy could be very different for different diseases • Gene transplantation • (to patient with gene deletion) • Gene correction • (To revert specific mutation in the gene of interest) • Gene augmentation • (to enhance expression of gene of interest)

10. What is Vector? • "Vector" is an agent that can carry a DNA fragment into a host cell.  • If it is used for reproducing the DNA fragment, it is called a "cloning vector".   • If it is used for expressing certain gene in the DNA fragment, it is called an "expression vector". • Commonly used vectors include plasmid, Lambda phage, cosmid and yeast artificial chromosome (YAC).

11. Type of vectors: • Plasmid • LambdaPhage • Cosmid • Fosmid • YAC • BAC • ViralVectors • Vectorsdesignedfor a certeinpurpose: • ExpressionVectors • PlantTransformationVectors • EpitopeTaggingVectors • Gene SilencingVectors • Vectorsystems: • ReporterSystems • SelectionSystems • InducibleSystems • RecombinaseBasedSystems • SpecialVectorSystems: • TopoVectors • TA Vectors • GatewaySystem • Transposons: • Retrotransposons • DNA transposons

12. Type of vectors: • Plasmid • LambdaPhage • Cosmid • Fosmid • YAC • BAC • ViralVectors

13. Plasmid • Plasmids are circular, indpendently replicating, double-stranded DNA, most often found in bacteria. They replicate quickly and are easily manipulated in the laboratory. Plasmids are typically 2-10 thousand base pairs in size (Corbley, 1999). While this small size allows plasmids the two aforementioned attributes, it aslo means that plasmids are limited in the DNA fragments they can clone. They are typically limited to fragments around 5 thousand base pairs (King, 2002).

14. Plasmid  Diagram of DNA sequence of a basic plasmid and incorporated construct.

16. ViralVectors • The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for clinically relevant gene therapy. • Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. This ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. • The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. • These are the major reasons why viral vectors derived from retroviruses,adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. 

17. RetroViralVectors • Retrovirus vectors represent the most prominent delivery system, since these vectors have high gene transfer efficiency and mediate high expression of therapeutic genes. • Efficient gene transduction and integration depend on the inclusion in the retroviral vector of a number of cis-acting viral elements. (1) a promoter and polyadenylation signal in the viral genome; (2) a viral packaging signal (ψ or E) to direct incorporation of vector RNA into virions; (3) signals required for reverse transcription, including a transfer RNA-binding site (PBS) and polypurine tract (PPT) for initiation of first- and second-strand DNA synthesis, and a repeated (R) region at both ends of the viral RNA required for transfer of DNA synthesis between templates; (4) short, partially inverted repeats located at the termini of the viral LTRs required for integration. An important general consideration in the design of retroviral vectors is the effect of viral replication on vector structure. After one round of viral replication, the U3 regions in both LTRs are derived from the U3 region originally present in the 3′LTR in the plasmid form of the vector, and both U5 regions are derived from the U5 region originally present in the 5′LTR in the plasmid. Ordinarily, R sequences should arise primarily from the 5′plasmid LTR, but they may also include 3′plasmid LTR sequences.

18. LentiViralVectors • Lentiviral vector constructs have proven to be very productive in terms of transduction due to their ability to infect both replicating and non-replicating cells, including stem cells. Lentiviralvectors are becoming the vectors of choice for short-interfering RNA (siRNA) delivery (Sachdeva et al., 2007). • The increased use of lentiviral vector constructs in established and novel research applications makes it essential for laboratory workers to understand and protect themselves from related exposure hazards.

19. AdenoViralVectors • Adenoviruses are medium-sized (90–100 nm), nonenvelopedicosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. There are over 51 different serotypes in humans, which are responsible for 5–10% of upper respiratory infections in children, and many infections in adults as well. When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated (transient) into the host cell's genetic material. The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribedjust like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene.

20. Vectorsystems: • ReporterSystems • SelectionSystems • InducibleSystems • RecombinaseBasedSystems

21. ReporterSystems • A gene consists of two functional parts: One is a DNA-sequence that gives the information about the protein that is produced (coding region). The other part is a specific DNA-sequence linked to the coding region; it regulates the transcription of the gene (promoter). The promoter is either activating or suppressing the expression of the gene. • The purpose of the reporter gene assay is to measure the regulatory potential of an unknown DNA-sequence. This can be done by linking a promoter sequence to an easily detectable reporter gene such as that encoding for the firefly luciferase. • Common reporter genes are β-galactosidase, β-glucuronidase and luciferase. Various detection methods are used to measure expressed reporter gene protein. These include luminescence, absorbance and fluorescence.

22. GFP

23. Luciferase

24. SelectionSystems • Selection markers are protein coding sequences that confer a selective advantage or disadvantage to host chassis. For example, a common type of prokaryotic selection marker is one that confers resistance to a particular antibiotic. Thus, cells that carry the selection marker can grow in media despite the presence of antibiotic. • Most plasmids contain antibiotic selection markers so that researchers can ensure that the plasmid is maintained during cell replication and division. (Cells that lose a copy of the plasmid will soon either die or fail to grow in media supplemented with antibiotic.) • A second common type of selection marker, often termed a positive selection marker, are those that are toxic to the cell. Positive selection markers are frequently used during cloning to select against cells transformed with the cloning vector and ensure that only cells transformed with a plasmid containing the insert.

25. Positive-NegativeSelection • A strategy used in making gene knockouts designed to enrich for homologous recombinants and select against random integration of the targeting vector. Cells transfected with the targeting vector incorporating two selectable markers are first selected for their resistance to an antibiotic such as G418 (positive selection) and then for the loss of a second marker, such as HSV thymidinekinase (HSVtk), which confers sensitivity to ganciclovir (negative selection). HSVtk placed at one end of the linear targeting vector is lost on homologous recombination but is retained on random integration.

26. Positive-negative selection using neomycin andthymidinekinase (TK) • What are G418 and ganciclovir used for? • The neomycin gene confers resistance to G418while TK renders cells susceptible to ganciclovir.Neomycin is used as a positive selection markerwhile TK is used as a negative selection marker. • Generally, they are used together in a strategy commonly referred to as ‘positive-negativeselection’.

27. Gene therapy In vivo Ex vivo

28. Different Delivery Systems are Available • In vivo versus ex vivo • In vivo = delivery of genes takes place in the body • Ex vivo = delivery takes place out of the body, and then cells are placed back into the body

29. EX VIVO IN VIVO in vivo and ex vivo schemes http://laxmi.nuc.ucla.edu:8237/M288/SChow_4_10/sld005.htm

30. In vivo gene therapy 1. The genetic material is transferred directly into the body of the patient 2. More or less random process; small ability to control; less manipulations 3. Only available option for tissues that can not be grown in vitro; or if grown cells can not be transferred back

31. Ex vivo gene therapy • The genetic material is first transferred • into the cells grown in vitro 2. Controlled process; Genetically altered cells are selected and expanded; more manipulations 3. Cells are then returned back to the patient

32. In vivo techniques usually utilize viral vectors • Virus = carrier of desired gene • Virus is usually “crippled” to disable its ability to cause disease • Viral methods have proved to be the most efficient to date • Many viral vectors can stable integrate the desired gene into the target cell’s genome

33. Side Effectscould be… Replication defective viruses adversely affect the virus’ normal ability to spread genes in the body • Reliant on diffusion and spread • Hampered by small intercellular spaces for transport • Restricted by viral-binding ligands on cell surface  therefore cannot advance far. • Retroviruses convey a risk of insertional mutagenesis

34. Gene Therapy Vectors Vectors deliver genes to cells Therapeutic gene (Transgene) Transcription Translation Therapeutic protein Vectorfor efficient gene delivery 34

35. Types of Gene Therapy Vectors Non-viral vectors Naked DNA Liposomes/DNA Polymer/DNA complex (polyplex) Liposome/Polymer/DNA (lipopolyplex) Viral vectors DNA viruses Adenovirus Herpes Simplex Virus RNA viruses Retrovirus 35

36. D. Limitations of Gene Therapy • Gene delivery • Limited tropism of viral vectors • Dependence on cell cycle by some viral vectors (i.e. mitosis required) • Duration of gene activity • Non-integrating delivery will be transient (transient expression) • Integrated delivery will be stable

37. Liposomes Next level idea – why naked DNA? Lets’ wrap it in something safe to increase transfection rate Lipids – are an obvious idea ! Therapeutic drugs

38. DNA delivery of genes by liposomes Cheaper than viruses No immune response Cytotoxicity 100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector

39. Gene Therapy Strategies Gene Replacement Replace ‘faulty’ genes with normal genes Corrects inherited genetic errors Provides a missing function Monogenic diseases e.g. cystic fibrosis, haemophilia, X-SCID Gene Addition Delivers genes to provide a new function Polygenic diseases e.g. cancer 39

40. PTQA April 2008 40

41. Retrovirus vector December 19, 2007 Boy gets leukaemia after gene treatment to cure ‘bubble baby syndrome’ • 3 year-old with X-linked severe combined immunodeficiency (X-SCID) - immune system fails to develop • Treated with genetically modified virus to correct the faulty DNA that causes X-SCID • Inserting the replacement DNA activated another gene that promotes cancer • Now an acknowledged risk of gene therapy Also seen in 4 / 11 patients in a French trial One has died while 3 are in remission

42. APPENDIX 6 - GENE THERAPY Facilities Documentation Labelling Training Aseptic processing Cleaning Storage Transport Waste Disposal Spillage 42

43. Facilities Gene therapy should not be manipulated in clinical areas Basic Principles - Containment - Knowledge/ understanding/skill - Validated procedures Persons handling the product should be masked and gloved All disposable equipment and materials used for prep & admin - handled as biohazardous Dedicated facilities required -ve pressure isolators or Class II BSC +ve pressure room or lobby Containment level > 2 43

44. Clean room suite designed to provide protection to the cleanroom

45. Aseptic Manipulation Doses Calculation/dilutions/multiple dilutions Needle stick injury risk Units Particle Units/ml (PU/ml) Plaque Forming Units/ml (PFU/ml) Infectious particle Units/ml (IU/ml) Gene Transfer Units/ml (GTU/ml) Stability Container compatibilities - Plastic/glass adhesion Expiry date - Time to administration from thawing 45

46. Decontamination Cleaning Virucidal detergents (validated against GT vectors) Cleaning Validation Specific Detection methods needed for viruses that are virus specific and highly sensitive Waste Disposal On site validated autoclave for re-usable equipment Inactivation on-site for Class 3 vectors Validated autoclave Incineration Disinfectant treatment 46

47. Spillage Specific to GT vector Spillage kit Contents ( gloves, masks, aprons, goggles, disposable shoe covers, virucidal detergents, absorbent material, disposable forceps & biohazard incineration bag) Positioned in all GT handling areas Notification to HSE Accidental Exposure 47

48. SOPs needed Safe handling & protection Storage Operators (Not pregnant, breastfeeding or immunosuppressed) Training Facilities Spillage, contamination & needle stick Waste disposal, cleaning and transport 48

49. Risk Assessment Assess each product individually Cytolytic viruses Non-cytolytic viruses Replication competent Replication deficient Class I, II or III 49