Gene Therapy for Diabetes

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it gives the detailed description of general gene therapy and also the gene therapy for diabetes.

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Gene Therapy :

Gene Therapy Under the guidance of Dr. S.K.Umadevi M.Pharm Ph D, Head of the department, Pharmaceutics, Rao’s college of pharmacy, Nellore. Presented by Mrs. P.Haritha Sunil M.Pharm 1 st year 1

Points to recollect:

Points to recollect Gene Genetic disorders Chromosomes Law of inheritance 2

GENES:

GENES Carried on chromosomes. Basic physical and functional units of heredity. Specific sequences of bases that encode instructions on how to make proteins. Why proteins? It’s the proteins that perform most life functions and even make up the majority of cellular structures. 3

Why genetic disorders?:

Why genetic disorders? When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result. Each of us carries about half a dozen defective genes. Most of us do not suffer any harmful effects from our defective genes because we carry two copies of nearly all the genes, one derived from the mother and the other from father. Except male sex chromosomes. Have one X and one Y so only each cell has only one copy of the genes on the chromosomes. 4

Chromosome:

Chromosome It is an organized structure of DNA and protein found in cells. It is a single piece of DNA containing many genes, regulatory elements and other nucleotide sequences. Also contains DNA bound proteins which controls its functions. 5

Law of inheritance:

Law of inheritance In the majority of cases, one normal gene is sufficient to avoid all the symptoms of the disease. If the potentially harmful gene is recessive then its normal counter part will carry out all the tasks assigned to both. Only if we inherit from our parents two copies of the same recessive genes will a disease develop. 6

What actually the gene therapy mean?:

What actually the gene therapy mean? It 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. 7

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Simply it is the technique for correcting defective genes that are responsible for disease development. It is used either to prevent a defective gene from producing its protein or to increase the concentration of the normal protein produced in the body by insertion of DNA or RNA fragments. In most gene therapy studies a normal gene is inserted into the genome to replace an abnormal disease causing gene. 8

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Although the technology is still in the infancy it has been used with some success. 9

How the genes can be transferred?:

How the genes can be transferred? A carrier called a vector is used to deliver the therapeutic gene to the patient’s target cells. C urrently the most common type of vectors are viruses that have been genetically altered to carry normal human DNA. V iruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. S cientists have tried to harness this ability by manipulating the viral genome to remove disease causing genes and insert therapeutic ones. T he vector then unloads its genetic material containing the therapeutic gene into the target cell. T he generation of a functional protein from the therapeutic gene restores the target cell to a normal state. 10

The Lytic Cycle:

The Lytic Cycle 11

Strategies in gene therapy:

Strategies in gene therapy Gene augmentation therapy: In this DNA is inserted into the genome to replace the missing gene product . Gene inhibition therapy: In this the antisense gene inhibits the expression of the dominant gene. 12

Approaches for gene therapy:

Approaches for gene therapy There are two approaches to achieve gene therapy. Somatic cell gene therapy: Uses the somatic cells of an organism. Eg; bone marrow cells, blood cells, skin cells, intestinal cells. This involves the insertion of a fully functional and expressible gene into a target somatic cell to correct a genetic disease permanently. Germ cell gene therapy: The reproductive cells of an organism constitute the germ cell line. For safety, ethical and technical reasons, it is not being attempted at present. All gene therapy to date on humans has been directed at somatic cells, where as the other type in humans remains controversial. Genetic alterations in somatic cells are not carried to the next generations. Therefore somatic cell gene therapy is preferred. 13

Diseases and their gene therapies:

Diseases and their gene therapies Disease Cystic fibrosis Thalassemia Sickle cell anemia Head and neck cancer Breast cancer AIDS Short stature * Diabetes * Phenylketonuria * Citrullinemia * Gene therapy Cystic fibrosis transmembrane regulator α or β globin β globin P 53 Multidrug resistance I rev and env Growth hormone Glucose transporter 2, glucokinase Phenylalanine hydroxylase Arginosuccinate synthetase * Mostly confined to animal experiments 14

Ex vivo gene therapy:

Ex vivo gene therapy Involves the transfer of genes into cultured cells which are then reintroduced into the patient. Can be applied to only selected tissues whose cells can be cultured in the laboratory. Eg Bone marrow cells. It 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 and grow. Transplant the modified cells to the patient. Uses patient’s own cells for culture and genetic correction. Thus it is not associated with adverse immunological responses after transplanting the cells. 15

Vectors used:

Vectors used Viruses Frequently used vectors, especially retrovirus. RNA is the genetic material. Enters the host cell and synthesizes DNA from RNA. This formed viral DNA (provirus) gets incorporated into the DNA of the host cell. Human artificial chromosome A synthetic chromosome that can replicate with other chromosomes besides encoding a human protein. Safe to use than the viruses. Bone marrow cells Contains totipotent embryonic stem cells. Capable of dividing and differentiating into various cell types (RBC, platelets, macrophages, osteoclasts, B and T lymphocytes). Hence most widely used technique. All the genetic disorders that respond to bone marrow transplantation are likely to respond to ex vivo gene therapy. 16

Examples :

Examples Therapy for adenosine deaminase deficiency. (ADA) Carried out to correct the deficiency of the enzyme ADA. Severe combined immunoefficiency (SCID) Rare inherited immune disorder associated with T and B Lymphocytes. 17

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In vivo gene therapy:

In vivo gene therapy Involves the direct delivery of the therapeutic gene into the target cells of a particular tissue of a patient. Liver, muscle, skin, spleen, lung, brain and blood cells. Can be carried out by viral or non viral vector systems. Success of the therapy depends on the Efficiency of the uptake of the therapeutic gene by the target cells. Intracellular degradation of the gene and its uptake by nucleus. The expression capability of the gene. Gene delivery by viruses Retro viruses Adenoviruses Adeno associated viruses Herpes simplex viruses By non viral systems Pure DNA constructs – directly into the target tissues. Lipoplexes – lipid DNA complexes that have DNA surrounded by lipid layers. Human artificial chromosomes – carries large DNA. 19

Diabetes mellitus:

Diabetes mellitus Most common endocrine disorder. Chronic condition. Characterized by hyperglycemia due to impaired insulin secretion with or without insulin resistance. 20

Types of diabetes according to their etiology:

Types of diabetes according to their etiology Type I β cell destruction Islet cell antibodies present Strong genetic link Onset below 30 yrs Faster onset of symptoms Insulin must be administered Not overweight Extreme hyperglycemia causes D. ketoacidosis. Type II No β cell destruction No Islet cell antibodies Very strong genetic link Onset above 40 yrs. Slower onset of symptoms Diet control and oral hypoglycemics are enough Overweight Extreme hyperglycemia causes hyperosmolar non ketotic hyperglycemia. 21

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Goal of diabetes therapy – to maintain normoglycemia irrespective of the diet intake. For more than 80 yrs insulin is the only treatment. Another option – whole pancreas or islet cell transplantation , but difficult and complex shortage of cadaveric pancreases immunosuppression Recent advancement – successful islet cell transplantation by glucocorticoid free immunosuppressive regimen Major goal in the treatment to generate an unlimited source of cells exhibiting glucose responsive insulin secretion. should be used for transplantation. ideally without the need for systemic immunosuppression. 22

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D iabetes C ontrol and C omplication T rial – tight glucose control is necessary to lower the incidence of diabetic complications. Can be done by multiple insulin injections. But more number of doses leads to hypoglycemic condition due to the absence of ideal glucose sensing system coupled to insulin administration Development of an artificial glucose sensor – substantial obstacles that need to be overcome. Thus, Cell transplantation therapy The best solution for the restoration of normal physiological glucose level. Gene therapy for diabetes 23

Cell transplantation therapy:

Cell transplantation therapy Hyper acute rejection due to the pre existing anti bodies to an α – galactosyl xeno epitope in pigs. Endogenous porcine retrovirus infections. so human beta cells are an attractive source but Limited availability so to be expanded in vitro in order to treat the large number of insulin dependent diabetic patients. 24

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PROBLEMS IN THE EXPANSION OF 10 β CELLS:

PROBLEMS IN THE EXPANSION OF 1 0 β CELLS Signals that trigger the β cell proliferation are incompletely understood Due to proliferation they tend to loose the differentiated function and have a very limited in vitro life span. Expansion is limited to 10 – 20 population doublings after which they undergo growth arrest due to cellular ageing. ANOTHER OPTION FOR TRANSPLANTATION Introduction of complex cellular machinery- responsible for the release of glucose responsive insulin from non β cells. Wide range of choice of starting cell No cellular machinery is able to mimic glucose responsive insulin secretion. So the best way is – human β cells or β cell precursors 26

β cell precursors:

β cell precursors Source of tissue for transplantation due to increased proliferative potential. Not yet been formally demonstrated. In vivo – neogenesis of endocrine islets from ductal epithelium has been described after various experimental conditions such as 90% pancreatectomy Pancreas wrapping in the rodent Or when transplanted together with fetal mesenchyme into mice. In vitro – neogenesis of endocrine islets from ducts with the use of matrix and growth factors. It is an approach to human islet propagation in order to increase the mass of endocrine tissue obtained from cadaveric pancreases. Recently shown – human ductal cells exposed to matrigel and growth factors could be directed to differentiate into endocrine islet cells in vitro. Another possible source Embryonic stem cells – have the ability to differentiate in vitro into different cell lineages. Insulin secreting cell clone from undifferentiated ES cells normalized the glycemia in streptozocin – induced diabetic mice 27

Gene transfer methods:

Gene transfer methods Gene delivery into 1 0 β cells has to meet the following requirements Expression of the trans genes within post mitotic cells. The potential need to target a specific cell type with in the islet. Should achieve an optimal duration of transgene expression. Methods used are Non viral methods Calcium phosphate co precipitation method Lipofection Direct micro injection Electroporation Biolistics Viral methods Retroviral Adenoviral Lentiviral 28

Non viral methods:

Non viral methods Calcium phosphate co precipitation method Simple and non expensive method for genetically modifying pancreatic cells. It can produce either transiently transfected cells or cells that able to stably express the transgene 29

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Lipofection The transfer of genes with the help of liposome is called Lipofection. These have been used as high efficiency transfection agents of cells both in vitro and in vivo unlike the above (In vitro) Advantage of in vivo – liposomes are directly injected into the blood stream and is less invasive than the other treatments like transplantation. Liposome containing DNA have minimal +ve charge which improve their interaction with target cells and the consequent transfection efficiency. 30

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Direct microinjection Directly injecting DNA into cells – an effective method Not suitable for targeting large number of cells – need to be targeted individually. 31

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Electroporation Creates permeable membrane for gene transfer by applying high voltages to cells. In many cases causes cell death. For efficient gene transfer into the islet cells – first the cells need to be dissociated from the clusters of cells into single cell suspensions. During dissociation – may be a change in their morphology and may become non functional. Though gene transfer is possible – cannot efficiently integrate DNA into the host genome. 32

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Biolistics use of a gene gun to transfect cells with a transgene. It rapidly discharges DNA – micro projectiles into cells. Produces high transfection efficiencies than others. 33

Viral methods:

Viral methods Choice of an appropriate vector requires careful consideration. To be a successful vector the following things to be satisfied. Simple to manufacture in large numbers. Have the ability to target to the specific site. Able to transduce both dividing and non dividing cells. Result in high transduction efficiency Should not illicit a strong immune response Allow for long term expression of the transgene. Retroviral vectors Retrovirus can efficiently adsorb onto and enter a target cell where the RNA of the virus is copied into DNA by reverse transcriptase enzyme. DNA thus formed gets incorporated into chromosomal DNA by host. The gene thus expressed is under the control of the virus. Retrovirus mediated gene transfer requires the division of target cells within a few hrs of vector entry.(disadv) Advancement – a new class of retroviral vectors based on lentivirus had been developed – overcomes the limitation. 34

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Adenoviral vectors Able to transduce both dividing and non dividing cells Can be of both human and non human type. Can be prepared in high titers. These viruses can infect insulin secreting cells and have been shown to be able to transduce rodent islets. Weakness – vector antigens show potent immune responses and the inserted DNA is episomal resulting in short term gene expression.(natural pathogen of humans). Lentiviral vectors Promising tool – targets quiescent and dividing cells Have similar characteristics to both retro and adeno viral vectors. Able to transduce primary and post mitotic cells such as neurons, liver, muscle cells, primary endothelial cells and islets. Transduces both dividing and non dividing cells and do not show any potent immune responses. Eg: HIV I virus. 35

Gene therapy treatments:

Gene therapy treatments Maintenance of euglycemia can be achieved by genetic manipulations. Ectopic gene expression Engineered islets Induction of immune tolerance Interference with antigen presentation 36

Ectopic gene expression:

Ectopic gene expression Expression of genes in cell types that are not their usual area of expression. Widely used technique – but the difficulty is requirement of immunosuppressive measures. Important features of β cells that are essential in the regulation of blood sugar levels include Continuous monitoring of glucose levels. Regulated transcription and translation of pro insulin. Regulated pro insulin processing to mature insulin. Regulated storage of mature insulin. Regulated secretion of mature insulin to a stimulus (glucose). Alternatives to β cells for manipulation into insulin producing cells Hepatocytes , fibroblasts Keratinocytes Neuroendocrine cells etc 37

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Hepatocytes – similar to β cells as they facilitate the removal of glucose from the blood stream by phosphorylation by glucokinase in response to high glucose levels. Good cells for genetic manipulation – essential regulators of carbohydrate metabolism via insulin. But not ideal – inability to store insulin and do not secrete molecules in response to glucose. Stem cells – used as surrogate β cells. Have the ability to multiply in culture, unlike islets. These are immune privilized so as to overcome the auto immune responses that destroys islets. 38

Engineered islets:

Engineered islets Transplantation of any organ or tissue other than auto grafts requires some protection from the immune system. It can be achieved by any of the following methods. Grafting into immunologically privilized sites (brain, thymus, testes). Isolation from the immune system(encapsulation) Prevent immune attack by altering the grafts by either Graft modification to prevent recognition of the graft as foreign. Graft modification to allow the graft to induce inactivation or apoptosis of the immune cells. Allotransplantation of human islets is one method of returning BGL to normal levels. Large quantities of functioning islets are required for improvement of T1D after transplantation. However the process of islet isolation and purification destroys many of the essential microvasculature for islet survival. 39

Induction of immune tolerance:

Induction of immune tolerance To prevent the immune system from recognizing and attacking our organs and cells, all T cells are screened in the thymus. Any T cells that reacts to presented self antigens are killed. When the screening mechanism fails T1D occurs – treatment for T1D is to induce tolerance to islets by introducing islets antigens to the thymus. This can be achieved by genetic manipulation of vectors to encode islet antigens, followed by injection into the thymus. 40

Interference with antigen presentation:

Interference with antigen presentation Antigen presenting cells (APCs), the dendritic cells (DCs) and macrophages are responsible for activating T cells by presentation of the correct antigen. Prevention of interactions between the APCs and T cells inhibits its activation and in T1D, it stops the destruction of insulin producing islets. Prevention of APC activation. Prevention of antigen processing by APCs. Reduction of interaction between islets and secreted molecules of APCs. Induction of T cell death via interaction with genetically induced expression. 41

Conclusion :

Conclusion Provided an opportunity to modify or replace genes that cause a disease. A more sophisticated vector design and improved understanding of gene mechanism is required. Not even a single gene transfer systems meet all the needs. Properties of non viral vectors are to be optimized. A better in vitro - in vivo correlation could be attained. An ex - vivo approach is adopted to introduce genes to the bone marrow cells. Just a technical extension of the present bone marrow transplantation techniques. 42

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