Establishing a Zinc Finger Nuclease Platform for Targeted Genome Editing in Human Cell Lines, Primary Keratinocyte Stem Cells and Cardiomyocytes in Vivo
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Hannover Medical School Institute of Experimental Hematology Establishing a zinc finger nuclease platform for targeted genome editing in human cell lines, primary keratinocyte stem cells and cardiomyocytes in vivo INAUGURAL - DISSERTATION For the degree of Doctor of Natural Sciences - Doctor rerum naturalium – (Dr. rer. nat) Submitted by Kafaitullah Khan Place of birth - Zhob, Pakistan Hannover 2012 Angenommen vom Senat der Medizinischen Hochschule Hannover am 23.05.2012 Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover Präsident: Prof. Dr. med. Dieter Bitter-Suermann Betreuer: Prof. Dr. phil. Toni Cathomen Kobetreuer: PD Dr. med Albert Heim 1. Gutachter: Prof. Dr. phil. Toni Cathomen 2. Gutachter: PD Dr. med Albert Heim 3. Gutachter: Prof. Dr. rer. nat. Jürgen Alves Tag der mündlichen Prüfung vor der Prüfungskommission: 23.05.2012 Prof. Dr. rer. nat. Jürgen Alves Prof. Dr. phil. Toni Cathomen PD Dr. med Albert Heim Prof. Dr. rer. nat. Jürgen Alves Dedicated to my grandmother Table of contents 3 Table of Contents 1. Introduction...........................................................................................................4 1.1. Genome editing ..............................................................................................4 1.1.1. Gene addition .........................................................................................5 1.1.2. Targeted gene addition...........................................................................5 1.1.3. Targeted gene correction........................................................................6 1.1.4. Targeted gene knockout or disruption ....................................................7 1.1.5. Chromosomal rearrangement.................................................................8 1.2. Nucleic acid delivery systems.........................................................................9 1.2.1. Non-viral delivery systems......................................................................9 1.2.1.1. Physical methods of nucleic acid delivery ....................................9 1.2.1.2. Chemical methods of nucleic acid delivery .................................11 1.2.2. Viral delivery systems ...........................................................................12 1.2.2.1. Retroviruses................................................................................13 1.2.2.2. Lentiviruses.................................................................................14 1.2.2.3. Adenovirus..................................................................................15 1.2.2.4. Adeno-associated virus...............................................................16 1.2.2.5. Other viral vectors.......................................................................19 1.3. Zinc finger nuclease......................................................................................20 2. Aims of the thesis ...............................................................................................22 3. Publication 1 .......................................................................................................23 4. Publication 2 .......................................................................................................33 5. Summary of results and discussion ....................................................................43 6. Summary ............................................................................................................53 7. Zusammenfassung .............................................................................................54 8. References .........................................................................................................55 9. Appendix.............................................................................................................75 9.1. List of own publication ..................................................................................75 9.2. Curriculum Vitae ...........................................................................................76 9.3. Acknowledgment ..........................................................................................77 9.4. List of abbreviation........................................................................................78 9.5. Declaration ...................................................................................................80 Introduction 4 1. Introduction Living organisms have a certain set of genes which are their unit of heredity. Genes can be defined as “a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions” [1]. Genes have information for certain type of structural or functional proteins. These proteins have a wide range of functions which includes defence against pathogen, homeostasis, maintenance and regulation of different cellular process. Most of diseases have a genetic factor which is either entirely or partially responsible for that disease. Disorders like thalassaemia, Sickle cell anaemia, and Gaucher’s have either completely lack or has mutation in a particular gene while diseases like hypertension and hypercholesterolemia involve interaction of genetic and environmental factors. In some other disorders, like Angelman syndrome, the causative agent is the loss of particular gene activity in specific cells. Even the infectious disease have genetic basis which involve the genes of the infectious agent and the host. Some of these genetic/infectious diseases have no treatment while in the case where treatment is present, majority of them are prophylactic rather than curative, by minimizing or reducing the severity of the diseases instead of providing permanent solution. One example of it is treatment of Gaucher's disease. Gaucher's disease is lysosomal storage disease due to accumulation of fatty substances in cells and certain organs [2]. The causative agent of this disease is a recessive mutation in a glucocerebrosidase gene which leads to the deficiency of the glucosylceramidase, an enzyme that degrades fatty acid glucosylceramide into D-glucose and N-acylsphingosine. Current treatment for Gaucher's disease includes enzyme replacement therapy with intravenous introduction of recombinant glucosylceramidase but this treatment is very expensive and should be continued for whole life. So to improve the life standard of these patients, there is a need of more effective and convenient long lasting therapy like gene based therapy or simply gene therapy. Gene-based therapy, which can be achieved by genome editing, are broadly define as “the introduction, using a vector, of nucleic acids into cells with the intention of altering gene expression to prevent, halt or reverse a pathological process” [3]. 1.1 Genome editing Depending upon the scientific or medical need editing of the genome can be performed by different ways including gene addition, targeted gene addition, targeted gene correction, targeted gene disruption and chromosomal rearrangements. Introduction 5 1.1.1 Gene addition Gene addition is the insertion of a functional copy of a mutated native gene. The first type of viruses used for gene addition therapy was retroviruses as their normal life cycle favour them to be used for permanent gene delivery in cells. The biggest barrier for the use of gene addition therapy is its inability to specify the insertion point of the transgene. This "stone throwing" approach of random insertion of a therapeutic gene, despite the successes of transferring it, may result in malignant transformation from the insertional activation of oncogenes [4]. In addition, randomly inserted genes are also subjected to silencing and position-effect variegation which make their expression unreliable and unpredictable [5, 6]. vice versa the randomly integrated transgene may affect endogenous genes and chromatin with the potential to change the cell behaviour and induce its transformation [7]. With some setbacks [8], gene addition has been used to successfully treat many diseases [9-12]. 1.1.2 Targeted gene addition Gene targeting is the method which allows for the precise and efficient modification of the genome. This method holds great potential not only as a tool for studying function of particular genes but also in biotechnology and human gene therapy [13]. Gene targeting or gene replacement strategy can be defined as “replacement of the endogenous DNA segment, with exogenously introduced DNA fragments, at predetermined genomic location by harnessing homologous recombination”. Targeted gene addition can be permanent or conditional. Conditional means that the transgene may express at specific time during the development of the organism or its expression may be specific to certain tissue. The expression of transgene, for example, can be made specific to a tissue or cells by using tissue specific promoter [14, 15] or detargeting by employing tissue specific microRNA (mir) target sites [16]. Targeted gene addition has three important advantages over other methods for gene editing. First it is possible to choose the genetic locus for gene editing as the addition takes place at predetermined genomic location. Second, the investigator can use full advantage of all the resources provided by the known sequences of the organism, especially for the mouse and human genomes. Third, investigators have control over the degree of modulation of the chosen genomic location