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GENE THERAPY Giri naramsetti Sri venkateswara college pharmacy Srikakulam Andhra University

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Eukaryotic cell

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A chromosome is an organized structure of DNA and protein found in cells It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.

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Deoxyribonucleic acid, DNA — is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms A gene is a unit of heredity in a living organism, that code for a type of protein, responsible for a specific character expression.

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Gene therapy is the insertion, alteration or removal of genes with in an individual cells and a biological tissue to treat disease. Gene therapy is “ the use of a gene as a medicine” involving the transfer of a therapeutic or working copy of a gene into specific cells of an individual in order to repair a faulty gene copy. DEFINITION

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Gene Therapy Mechanisms

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Correct a mutated gene (video 1)

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Replacement of defective or mutated gene (video) Insertion of new functional gene: Video 2 Insertion of missing gene.

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Block the target gene expression . Protein synthesis is blocked, in turn the expression or action of the viral gene cannot be carried out.

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Interferes with life cycle of infectious diseases (video). Interferes with life cycle of infectious disease. Interfere with the life cycle of the infectious diseases Video 3 INTERFERE WITH VIRAL GENOME

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Germ line gene therapy Germs cells, i.e sperms or eggs, are modified by the introduction of functional gene. Change is inherited. + Fertilization

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Somatic gene therapy Therapeutic gene are transferred Into the somatic cells ( diploid, any biological cell forming the body or organ as directed) . Modification is restricted.

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Medium for cell and viral cultures: ions; Na, K, Ca, Mg, Cl, P, bicarbonate. • Trace elements; iron, zinc, selenium. • Sugars such as glucose, vitamins. • Amino acids (lysine,valine, leucine etc) • Serum; contains hormones, growth factors to promote proper growth. Dialyzed serum. Basic media: Overall process of water purification • Reverse osmosis or distillation (remove major chemicals) • Charcoal filtration (↓organic and inorganic impurities) • Deionization (↓trace metals or ions) • Micropore filtration (↓microbial contamination) Water: BME (Eagle’s Basal Media) EMEM (Eagle’s Minimum Essential Media) GMEM (Glasgow’s Modification of Eagle’s medium) DMEM (Dulbecco’s Modification of Eagle’s Medium) RPMI (Roswell Park Memorial Institute medium) Different culture media:

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2. Transduction: Transduction refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transfection: Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral method in eukaryotic cells.

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3.Transformation: Transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surrounding and taken up through the cell membrane.

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Vectors Non – immunogenic Stable in blood stream. Protect DNA during transport. Small enough to extravagate. Cell and tissue specificity. Bio – compatible. Ideal characters of a carriers or vectors Vehicle used to transfer or carry genetic material to a target cell.

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DIFFERENT VECORS IN USE: Viral vectors Non- Viral vectors Chemical vectors Nano particles Hybrid vectors Nanorobots

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Viral vectors

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Adeno virus A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans.

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Adeno-associated virus A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19 (pancreatic region)

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A class of double-stranded DNA viruses that infect a particular cell type, neurons.

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Envelop protein pseudo typing of viralvectors Cis-trans acting elements Attachment to and entry into a susceptible cell is mediated by the protein envelope on the surface of a virus. Retro virus have limited natural host cell ranges, and although adenovirus and adeno-associated virus are able to infect a relatively broader range of cells efficiently, some cell types are refractory to infection by these viruses as well Transfer construct; this carries gene and necessary viral genome. Which helps in packing sequence , repeats for replication, priming of reverse transcription. Cis- same piece of dna carries viral genome and gene of interest. Trans- will be on different dna strands.

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Retro virus A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cell.

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Chemical methods

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Oligonucleotides The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease process. The transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression. Uses small molecules of RNA called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. Single stranded DNA oligonucleotides have been used to direct a single base change within a mutant gene.

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Dendrimers A dendrimer is a highly branched macromolecule with a spherical shape. Positive surface charged dendrimer and negatively charged DNA OR RNA. Charge complimentarily leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching the destination the dendrimer-nucleic acid complex is then taken into cell via Endocytosis.

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Nano particles

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lipoplexes Most common use of lipoplexes has been in gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes. Condensation of DNA (-ve charged) with lipids (+ve charged) to facilitate to encapsulation of Dna into liposome. + Use of helper lipids called Electro neutral lipids, DOPE. LIPOPLEXES These have higher transfection efficiency, Endocytosis is means of uptake by the cell membrane.

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Gold nano particles This consists of nano sized gold particles surrounded with the specific antibodies and desired DNA molecules around it.

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Gold nano particles in body Gold nanoparticles delivery (video 5)

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Specialized method

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Virosomes These are hybrid vectors. Combination of Liposomes + inactivated HIV or Influenza virus. These have more efficient transfer in respiratory epithelial cell than either viral or liposomal methods alone. +

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Non-viral vectors

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Naked plasmids A plasmid is a double stranded DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA.

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Video 6

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Gene gun Under certain conditions, DNA (or RNA) become “sticky,” adhering to biologically inert particles such as metal atoms (usually tungsten or gold). By accelerating this DNA-particle complex in a partial vacuum and placing the target tissue within the acceleration path, DNA is effectively introduced . The cells that take up the desired DNA, identified through the use of a marker gene

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Different methods have been used to accelerate the particles: these include pneumatic devices; instruments utilizing a mechanical impulse or macroprojectile magnetic or electrostatic forces; spray or vaccination guns

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Sonoporation The transient permeabilization of cell membrane. High-amplitude acoustic waves can spontaneously nucleate and excite dissolved gas and vaporize liquid, forming cavitations gas bubbles in tissue. When bubbles are in the vicinity of solid boundaries (e.g. cell membranes), the collapse will be asymmetrical and can result in the formation of liquid microjets travelling at sonic speed toward the surface. Rise in temperature influences the fluidity of phospholipid bilayer membranes, cell membrane permeability could be changed directly as a consequence of the increased bilayer fluidity After entering the membrane the shell of drug delivery systems is disrupted and thereby the enclosed gene are released.

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Magnetofection The magnetic nanoparticles are made of iron oxide, which is fully biodegradable. Their association with the gene vectors (DNA, siRNA, ODN, virus, etc.) is achieved by (i) Salt-induced colloidal aggregation and (ii) Electrostatic interaction. The cellular uptake of the genetic material is accomplished by endocytosis and pinocytosis Principle is to concentrate the nucleic acid associated cationic magnetic nanoparticles to the cells by an appropriate magnetic field.

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Electroporation When a cell is exposed to an electric field, a transmembrane voltage is induced on the membrane, this leads to a significant increase of the electric conductivity and the permeability of the membrane. State of increased permeability of the membrane caused by an exposure to the electric field is called electroporation. Application of electroporation for transfer of DNA molecules into the cell, often referred to as electrogenetransfection When DNA Injection into the muscle is combined with electroporation, the gene expression is increased by two or three orders of magnitude.

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Nanorobots A nanorobot is a tiny machine designed to perform a specific task or tasks repeatedly and with precision at nanoscale dimensions. Nano robots work is mainly based on: Removal of the defective gene. Triggering off the main mutated or defective gene expression which causes the complications. Insertion or removal of gene at germ cell level. Nanotech robots deliver gene therapy through blood.

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Gene repair using nano robot (video) DNA REPAIR BY NANO ROBOT: Video 7

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Single gene defects Cystic fibrosis Muscular dystrophy Leber retinal amaurosis Sickle cell anemia Haemophilia Multi gene defects Asthma Arthritis Cancer Diabetics AIDS

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Cystic fibrosis Refers to the characteristic scarring (fibrosis) and cyst formation within the Lungs, GIT and Pancreas.

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Genes related (video) CFTR regulates the passage of the salts (NACL) across the membrane. Defective gene = cystic fibrosis transmembrane conductance regulator (CFTR). Defective chloride channels generally leads to mucus formation likely because of dehydration of the fluid surrounding affected cells.

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Therapy Defective CFTR gene was replaced by Normal functional gene. In the case of CF, gene therapy involves inhaling a spray that delivers normal DNA to the lungs. Vectors mostly used for this type of delivery include: ADENO VECTORS. ADENO ASSOCIATED VECTORS.

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Muscular dystrophy Refers to a group of hereditary muscle disease that weakens the muscle that move the body, due to lack of muscle proteins. Main cause is inability to properly create the protein dystrophin. Dystrophin protein is found in muscle fibre membrane, it acts like a massive spring, it joins the membrane actin filaments.

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The X-linked dystrophy gene (DMD) is by far the largest of 30,000 genes that encode. Its 79 exons cover 2.6 million base pairs. The size of dystrophin gene has been major challenge, The delivery of functional mini and micro dystrophins by recombinant adeno-associated virus (rAAV) Therapeutic anti-sense-induced exon skipping. Dystrophin replacement by utropin up regulation. That is to replace a defective gene or artificial dystropin Cdna construct into the nuclei of muscle cells.

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New methods: The delivery of functional mini and micro dystrophins by recombinant adeno-associated virus (rAAV) Therapeutic anti-sense-induced exon skipping. Dystrophin replacement by utropin upregulation

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So to deliver large dystropin large dystropin Cdna vectors with a large capacity were needed. High capacity gutless vectors from which all adenoviral genes were removed by passes this restriction and deliver extra benefits in the form of reduced host immune response. (ii) Herpes simplex virus carry large inserts. (iii) Full length dystropin Cdna is not a problem for non-viral Dna plasmid that can be engineered to contains large inserts. These vectors are synthetic and non-infectious; Electroporation, pressurized isolated limb perfusion (or) micro bubbles and ultrasound are needed to enhance transfection efficiency. VECTORS:

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Leber retinal amaurosis This is a condition ,which prevents the retina from processing light. In other words we can also say retina has no effect, that is contraction and relaxation procedure are completely absent. So final result is inability of the retina to send the signal to the medullary region, thus results improper or no recognition of the object or picture

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GUCY2D. CRB1. RPE65. RPGRIP1. AIPL1. TULP1. CRX. RDH1LCA13) CEP290 (LCA10) . IMPDH1 (LCA11) LCA5 (LCA5) CRX (LCA7) Autosomal dominant retinitis pigmentosa Night blindness Nystagmus Photophobia Severe infantile-onset cone-rod dystrophy Genes related: Mutations in these three genes are mainly responsible for improper growth and function of retina in particular.

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Vectors mainly used in this therapy are: 1.Adeno virus and 2. Retro virus.

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Sickle cell anemia Conversion of normal bi concave shaped red blood cells in to sickle shaped due to mutations in the genetic material..

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EFFECTS: Low oxygen carrying capacity by sickle shaped cell. Blockage of the small blood vessels due to its improper shape.

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The gene related to sickle cell anemia is the hemoglobin gene (HBB). The HBB protein is 146 amino acids long. The HBB gene is found on chromosome 11. Hemoglobin Gene video

The Proteins of Hemoglobin A : 

The Proteins of Hemoglobin A There are 4 protein subunits of Hemoglobin A An S mutation in one copy of the hemoglobin beta gene. carrier for Sickle Cell SICKLE CELL DISEASE

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Sickle cell genes An S mutation in one copy of the hemoglobin beta gene causes the sickle shaped blood and also low oxygen carrying capacity. The mutation that causes this change in the beta happens because a glutamic acid residue replaces a lysine residue at the sixth position of the beta globin chain.

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Two methods of gene therapy are being explored. Correction of Gene Turning Off Gene 1.Correction of Gene Scientists are looking at whether correcting the defective gene in sickle cell anemia and inserting it into the bone marrow of people with sickle cell anemia will result in the production of normal adult hemoglobin. 2.Turning Off Gene Some other scientists are looking at the possibility of turning off the defective gene and simultaneously reactivating another gene that turns on the production of fetal hemoglobin.

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Hemophilia A group of inherited bleeding disorders in which the ability of blood to clot is impaired.

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Normal clotting VIDEO 12

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Types: Hemophilia A, due to viii gene. Hemophilia B, due to ix gene. Hemophilic condition video 13

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Treatment The F VIII gene codes for the F VIII protein, this protein consists of 3 types of domains: The triplet A-domain of 330-380 amino acids. A unique B- domain of 908 amino acids. and duplicated c-domain of 160 amino acids. Sequence--- A1- A2- B- A3 C1- C2 A for cation binding C for phospholipid and binding. The B-domain deleted F VIII Cdna had shown to express a higher levels of FVIII protein as compared to full length. F IX gene for F IX protein. MoMLV based retro viral vectors. Lenti-viral vectors. Adeno viral vectors. Adeno-associated vectors. Vectors:

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Video 14 Asthma:

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Treatment: (i) GSTM 1 (vi) LTA (xi) STAT6 (xvi) IL4 (xxi) HLA-DQB1 (ii) IL 10 (vii) GRPA (xii) NOS1 (xvii) IL 13 (xxii) TNF (iii) CTLA-4 (viii) NOD 1 (xiii) CCL5 (xviii) FCER IB (xxiii) FCERIB (iv) SPINK 5 (ix) CC16 (xiv) TBXA2R (xix) ADRB2 (xxiv) IL4R (v) LTC 4S (x) GSTP 1 (xv) TGFB1 (xx) HLA-DRB1 (xv) ADAM33 VECTORS: Liposomes, Adeno virus, plasmids. The entry of this type of gene is mainly by inhalation mechanism which gives more promising entry through pulmonary region.

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Arthritis Inflammation and degradation of the fluid and bone due to the effected gene.

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video Video 15

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Gene & Vectors: Ex Vivo Retro virus - IL-1Ra TNFR: F c SOD IL – IRAcP IN Vivo Adeno virus – IL- Ira IL – 4 IL - 13 Thrombospodin 1 TLA – 4Ig IL – 1813P lenti virus : Endostatin Plasmid : HSV TK PLUS GANCICLOVIR

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VIDEO Cancers Video 16 Uncontrolled proliferation, that is division of the cell both in shape and its size . Mainly 2 types of tumors BEGNIN TUMOR METASTIC TUMOR

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This condition is due to decreased levels of insulin secretion by the pancreas. The increased sugar glucose levels in the blood due to improper tissue distribution of glucose. Diabetics

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Video 17 Normal Glucose management:

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Video 18 Types: Type 1 diabetics, beta cells of pancreas fail to produce enough insulin. Type 2 diabetics, cells produce enough insulin but cells become resistant to its effects. Hyperglycemia:

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Treatment: Hepatocyte nuclear factor (HNF)-1α. glucokinase gene HNF-4α, HNF-1β, insulin promoter factor (IPF)-1, and NeuroD1. GENES: VECTORS: Mainly Adeno- associated vectors is used to carry gene to the pancreatic level cells.

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AIDS: Video 19

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Treatment: Using Sirna or modified DNA to stop viral translation

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Some of the problems of gene therapy include: Multigene disorders – Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy. Problems with viral vectors – Viruses, the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient —toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.

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Immune response – Anytime a foreign object is introduced into human tissues, the immune system has evolved to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a possibility. Furthermore, the immune system's enhanced response to invaders that it has seen before makes it difficult for gene therapy to be repeated in patients. Short-lived nature of gene therapy – Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.

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Gene therapy promises to be the next great frontier of medical therapeutics. But after 15 - 20 years of intense research, that promise remains largely elusive. CONCLUSION: Promise for many inborn errors of metabolism. Vectors for controlling the expression of therapeutic genes. Methods for delivering vectors safely to patients.

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REFERENCES: Christopher H. Evans, Steven C. Ghivizzani, and Paul D. Robbins, GENE THERAPY FOR ARTHIRITIS. Rita Mulherkar, GENE THERAPY FOR CANCER CELLS. Jens Kurreck, ANTI SENSE TECHNOLOGY Mehboob A Hussain, Neil D Theise, GENE THERAPY FOR DIABETICS Tim W. R. LEE, David A. MATTHEWS and G. Eric BLAIR, GENE THERAPY FOR CYSTIC FIBROSIS Marinee K. L. Chuah, Desire Collen, Thierry VandenDriessche GENE THERAPY FOR HAEMOPHILIA. Judith C. T. van Deutekom and Gert-Jan B. van Ommen, MUSCULAR DYSTROPHY. Thomas W. Chalberg,Alexander Vankov,Fanni E. Molnar,Alexander F. Butterwick,Philip Huie,Michele P. Calos,and Daniel V. PalankerFOR RETINAL THERAPY

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