Gene therapy
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The sequencing of the human genome has been compared to putting a man on the moon, and it will certainly change health care, but the most important work lies ahead, in determining how to put the......

The sequencing of the human genome has been compared to putting a man on the moon, and it will certainly change health care, but the most important work lies ahead, in determining how to put the information to medical use. In this context, applications such as gene therapy are being explored. What was once seen as a science fiction dream is now becoming a real possibility.
Gene therapy is a new form of drug delivery that leads the patient's own cells to produce a therapeutic agent. It could potentially eliminate the need for repeated administration of proteins or drugs. Applications of gene therapy not only include rare inherited diseases but extend to common acquired disorders, including tumours (predominantly malignant melanoma) and haematological disorders, cardiovascular disease, and the acquired immunodeficiency syndrome. Gene therapy therefore could be a key element of medical practice in the future. Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, and hereditary diseases in which a defective mutant allele is replaced with a functional one. Although the technology is still in its infancy, it has been used with some success. Antisense therapy is not strictly a form of gene therapy, but is a genetically-mediated therapy and is often considered together with other methods.

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  • 1. Gene Therapy- Types, Methods, Vectors, Major Development, Problems Sanju Kumari
  • 2. • Gene therapy is the insertion of genes into an individual cells and tissues to treat a disease in which a defective mutant allele is replaced with a functional one• DNA is used as a therapeutic agent• Genetic diseases, hematological disorders, acquired immunodeficiency syndromes, cancers are mainly treated
  • 3. Types of gene therapy Germ line gene therapy Somatic cell gene therapy
  • 4. Somatic cell gene therapy IN VIVO EX VIVO
  • 5. Hematopoetic stem cell gene therapy
  • 6. Embryonic stem cell gene therapy • Neuronal degeneration results into retinis pigmentosa, retinal detachment,glaucoma • Neural stem cell may help to restore vision in patient • Epidermal growth factor or fibroblast growth factor used for proliferation and maintenance invitro
  • 7. Vectors for gene therapy• Viral vectors • Non-Viral vectors Retroviral vectors Oligonucleotides Lentiviral vectors Naked-DNA Adeno associated viral Polyplexes vectors Lipoplexes Adenoviral Vector Liposomes
  • 8. RNA Virus • Retrovirus -The enveloped virus particle contains two copies of the viral RNA genome, surrounded by a cone-shaped core • It is Integrating virus
  • 9. Retroviral life cycle
  • 10. • Separation of the cis and trans functions of a retrovirus in a recombinant, replication defective vector system• Recombinant virus is made by introducing all these elements into the same cell• SCID-X gene therapy
  • 11. Production of Retroviral particle (Verma & Weitzman, 2005)
  • 12. • Lentivirus – It encodes three to six additional viral proteins, which contribute to virus replication and persistence of infection• Human immunodeficiency virus type 1 (HIV-1) - based vectors most extensively studied• Thalassemia treatment
  • 13. DNA Virus • Adenovirus – double stranded linear DNA • Cause respiratory (especially common cold), intestinal and eye infections in human • It is non integrating virus • Used for cancer therapy
  • 14. Adenovirus
  • 15. Adenoviral-mediated Rybp expression promotes tumorcell-specific apoptosis • Rybp kills tumors but not non transformed cell • Ad-Rybp treatment result in Cytotoxic effect in the osteosarcoma cell line U20S • It enhances Fas-induced apoptosis • Ad-Rybp infection sensitizes cells to TNF-α treatment
  • 16. Ad-Rybp infection induces apoptosis
  • 17. • Adeno - associated virus Single stranded DNA Episomal forms Deliver genes to brain, muscle, eye
  • 18. • Treatment of hemophilia B• Deficiency of factor IX• Therapeutic delivery of the canine factor IX gene to hemophilic dogs.• An efficient, non- immunogenic and persistent vector for mediating therapeutic gene delivery
  • 19. Liposome • Liposomes are phospholipid vesicles (50 – 100 nm) • They have a bilayer membrane structure similar to that of biological membranes and an internal aqueous phase • Liposomes show excellent circulation, penetration and diffusion properties
  • 20. Lipoplexes and polyplexes • Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposome • When the organized structure is complexed with DNA it is called a lipoplex • Complexes of polymers with DNA are called polyplexes
  • 21. Lipoplexes and polyplexes mediated transfection
  • 22. Dendrimer • These are highly branched synthetic polymers (<15 nm) • It show layered architectures constituted of a central core, an internal region and numerous terminal groups • Wide application in Drug Delivery System (DDS) and gene delivery
  • 23. Dendrimer delivery of an anti-VEGF oligonucleotide intothe eye • Lipophilic amino - acid dendrimer is used to deliver an anti -vascular endothelial growth factor (VEGF) oligonucleotide (ODN-1) into the eyes of rats (Marano et al., 2005) • It inhibit laser - induced choroidal neovascularization (CNV) Dendrimer used for ODN-1 delivery
  • 24. Methods of gene transfer• Physical methods :- Microinjection Electroporation• Chemical methods:- Calcium phosphate precipitation
  • 25. Femtosecond laser-assisted microinjection into livingneurons • Femtosecond laser is used to perforate vital cells • Transfection efficiency reached almost 100%. • Advantage –  contact-free  non-disruptive  stable transfection.
  • 26. Optical damage of astrocyte irradiated by femtosecond laser
  • 27. Electroporation • Electroporation is the best non viral transfection technique in human endothelial and smooth muscle cells • High efficiency and acceptable survival rate • Require special buffer and programs • A range of voltage,capacitance and resistance settings is used • High number of cells and high plasmid amounts required is a weakness (Iversen,2005)
  • 28. Calcium phosphate transfection • Most popular tools in neuroscience research • Low cell toxicity and easiness to use Fluorescent images of GFP-transfected cells
  • 29. Chimeraplast (Richardson,2002)
  • 30. Triplex forming oligonuleotide (Richardson,2002)
  • 31. Small fragment homologus replacement (Richardson,2002)
  • 32. • Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells• Alternative approach to ‘cure’ genetic disorders caused by mutations• Establishement of MSCs cell lines with stably integrated mutant neomycin resistance and enhanced green fluorescent protein reporter genes• The genetically modified MSCs were able to engraft into many tissues of unconditioned transgenic mice (Flagler,2008)
  • 33. Sleeping beauty transposon system • Transposon- used as therapeutic agent for gene transfer • It has been used to accomplish stable chromosomal integration of functionoing gene in somatic cells of adult mice of the factor IX for hemophilia (Richardson,2002)
  • 34. • Somatic integration and long-term transgene expression in normal and hemophilic mice using a DNA transposon system• Sleeping beauty transposase efficiently insert DNA into the genomes of adult mammals using naked DNA• Long-term expression of human blood coagulation factor IX• Therapeutic in a mouse model of Haemophilia B (Yant,2000)
  • 35. Vectors for Sleeping Genetic assay forBeauty-mediated transgene integration intransposition cultured cells.
  • 36. Genetic assay used to recover transposons frommouse chromosomes
  • 37. Octaarginine-modified multifunctional envelope-typenanoparticles for gene delivery • Multifunctional envelope-type nanodevice (MEND) that mimics an envelope-type virus based on a novel packaging strategy. • DNA core packaged into a lipid envelope modified with an octaarginine peptide • Topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) • Type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo • Superior non-viral gene delivery system (Khalil et al.,2007)
  • 38. The multifunctional Hair follicle formation in miceenvelope type nano skin treated with MEND3device
  • 39. Major Development First Approved Gene Therapy Ashanthi De Silva - A rare genetic disease called severe combined immunodeficiency (SCID) Defective adenosine deaminase gene results in deficiency of ADA protein It plays important role in deamination reaction Lack of healthy immune system Dr. W. French Anderson with four-year old Ashanthi De Silva at U.S. National Institutes of Health
  • 40. • Correction of SCID(X) by exvivo gene therapy• Mutation in the gene encoding the common γc chain• γc chain is an essential component of five cytokine receptors, which are necessary for the development of T cells and natural killer cells.• CD34+ bone marrow cells from five boys are transduced ex vivo• Retroviral vector (Abina et al.,2002)
  • 41. Gene correction using retroviral vectors
  • 42. No. of cells after gene Normal sized thymustransfer
  • 43. Repeat administration of DNA / liposomes to the nasalepithelium of patients with cystic fibrosis • CFTR is a cAMP-activated chloride channel in the apical membrane of epithelial cells • Loss of CFTR leads to impaired electrolyte movement in the organs • CFTR cDNA complexed with DC-Chol/DOPE cationic liposomes (Hyde et al.,2000)
  • 44. CFTR immunohistochemical staining of nasal brushing cells
  • 45. Third strand-mediated psoralen-induced correctionof the sickle cell mutation • Plasmid containing a b-globin gene fragment transfected into cells Interaction of the psoralen delivery strand with the target sequence
  • 46. Screening assay for mutation correction (Varganov et al., 2007)
  • 47. Gene Therapy May Switch off Huntington Disease • RNA interference or gene silencing may be a new way to treat Huntingtons • siRNA is designed to match the RNA copied from a faulty gene • HD results from polyglutamine repeat expansion (CAG codon) in exon of huntingtin gene • Toxic gain of function on the huntingtin protein (Harper et al.,2005)
  • 48. RNAi expression toHuman htt expression by mouse striatumRNAi
  • 49. Gene Therapy Tackles Blood Disorder • Blood disorder Thalassemia • Additional splice site is formed due to mutation • Repairs errors in messenger RNA derived from defective genes Correction of aberrant splicing by modified U7 snRNAs
  • 50. Correction of aberrant splicing by U7.623 snRNA in -globin IVS2mutant cells (Vacek et al., 2003)
  • 51. Dystrophin expression in mice by intravascular injection ofnaked DNADuchenne muscular dystrophy (DMD) - lethal, X-linked,recessive disease caused by a defect in the dystrophingene Injection of DNA solution by tail Effect of histamine on gene artery and tail vein expression
  • 52. Gene Therapy for Parkinsons disease • Liposomes coated in a polymer call polyethylene glycol (PEG) • Viral vectors are too big to get across the "blood-brain barrier" This method has potential for treating Parkinsons disease Loss of dopaminergic neurons • Tyrosine hydroxylase gene therapy • Episomal based gene therapy (Pardridge et al., 2005)
  • 53. A super-coiled expression plasmid DNA encapsulated in an 85 nmpegylated PIL targeted to a cell membrane receptor (R) with areceptor-specific, endocytosing mAb (Pardridge et al.,2005)
  • 54. First Deafness Cure in guinea pig• Destruction of the hair cells in the cochlea that translate sound vibrations into nerve signals• A gene, called Atoh1, which stimulates the hair cells growth, was delivered to the cochlea by an adenovirus• Regained up to 80% of their original hearing thresholds (Kawamoto et al.,2005)
  • 55. Gene Therapy for Advanced Melanoma • Reengineer lymphocytes for expression of TCR to target and attack cancer cells in patients with advanced metastatic melanoma • Retroviral vector was constructed encoding the pmel-1 TCR genes targeting the B16 melanoma antigen, gp100 • Adoptive cell transfer • Transduction of C57BL/6 lymphocytes resulted in efficient pmel-1 TCR expression
  • 56. • Objective - The most severe forms of inherited blindness are collectively known as Leber congenital amaurosis (LCA) and are the first type of inherited blindness to be treated by gene therapy
  • 57. Materials and Methods • A recombinant AAV-2 vector was used to deliver a human RPE65 cDNA to the target retinal pigment epithelium cells • Maguire et al.used an AAV-2 vector with a constitutive promoter to drive transgene expression • Bainbridge et al.used elements of the endogenous RPE65 promoter • detected a progressive improvement in retinal sensitivity in one patient using both microperimetry and dark-adapted perimetry
  • 58. Results
  • 59. Assesment of visual mobility
  • 60. Problems in gene therapy • Short-lived nature of gene therapy • Immune response and toxicity • Problems with viral vectors • Restricted targeting of specific cell types • Multigene disorders • Insertional mutagenesis • Religious concern • Deaths may occured
  • 61. Failures of Viral Mediated Gene Therapy • Retroviral vector • Dr. Alan Fischer – Conducting gene therapy on SCID-X1 linked hereditary disorder • Hematopoietic stem cells from patients were stimulated and transduced ex vivo with MLV-based retroviral vector • Expressing the γc cytokine receptor subunit, and then were reinfused into the patients • During a 10-month follow up, γ c-expressing T and NK cells counts and function were comparable to age-matched controls • Two of the children developed T-cell leukemia
  • 62.  Adeno-Associated Virus Vector • Patients suffering from hemophilia B were treated with AAV vectors expressing human factor IX • Intramuscular injecting AAV factor IX vectors directly into liver, which in turn have shown some unexplained toxicity  University of Pennsylvania • A human Phase I clinical trial for ornithine transcarbamylase deficiencies • This trial was designed to test the safety of an E1/E4- deleted recombinant adenovirus vector • Jessie Gelsinger received highest dose and first person to die as result of vector delivery