1. Madiha Khalid 07-arid-1610

2.  Use of DNA as a pharmaceutical agent to treat disease.  The most common form involves DNA that encodes a functional, therapeutic gene to replace a mutated gene.  Other forms involve using DNA that encodes a therapeutic protein drug.  Once inside, the DNA becomes expressed by the cell machinery, resulting in the production of therapeutic protein, which in turn treats the patient's disease.

3.  A vector delivers a therapeutic gene into patient’s target cell.  Functional proteins are created from therapeutic gene causing cells to return to a normal state.

4. Gene therapy may be classified into the two following types  Somatic gene therapy In somatic gene therapy, the therapeutic genes are transferred into the somatic cells (non sex-cells), or body, of a patient.  Germ line gene therapy In germ line gene therapy, germ cells(sperm or eggs), are modified by the introduction of functional genes, which are integrated into their genomes.

5.  Ex vivo: where cells are modified outside the body and then transplanted again.

7.  Cardiovascular disease remains the leading cause of morbidity and mortality in developed countries.  The emergence of human gene therapy in the early 1990s led to numerous attempts, both experimental and clinical, to treat cardiovascular disease with gene therapy strategies.  Gene therapy holds considerable promise for the treatment of cardiovascular disease and may provide novel therapeutic solutions for both genetic disorders and acquired pathophysiologies such as arteriosclerosis and heart failure

8.  Recombinant DNA technology and the sequencing of the human genome have made candidate therapeutic genes available for cardiovascular diseases.  However, progress in the field of gene therapy for cardiovascular disease has been modest.  one of the key reasons is the lack of gene delivery systems for localizing gene therapy to specific sites to optimize transgene expression and efficacy.  Because cardiovascular disease is characteristically localized, the site-specific targeting of gene therapy for the cardiovascular system is necessary.

9.  For efficient delivery of therapeutic genes to the cardiovascular system, a series of barriers have to be overcome.  The gene vectors need to pass through the endothelial barriers in capillary walls when systemically injected.  Plasmid faces a threat of being degraded rapidly by the immune system or DNAse in serum before transfection.

10.  Viral gene vectors need to avoid the immunoreaction in circulation and transduction of non-target organs, mainly liver and spleen.  The plasmid needs to avoid being entrapped into lysosome or the endosome, where it will be degraded.  The gene vector has to penetrate the nuclear membrane to achieve the goal of gene therapy.

11.  A variety of cardiovascular therapeutic gene constructs have been studied in vitro and in vivo.  These constructs can be categorized into several groups  The tumor-suppressor p53  Metalloprotease inhibitor 3  hepatocyte growth factor  superoxide dismutase [SOD]  sarcoplasmic/endoplasmic reticulum calcium ATPase 2

12.  Mostly 2 types of vectors are used  Non-viral gene vectors  Plasmid DNA  Antisense and decoy RNA  Viral gene verctors  Retroviruses and lentiviruses  Adenoviruses  Adeno-associated viruses

13.  Low toxicity,  Considered to be the safest choice for therapeutic gene transfer  Unfortunately, the inherently low expression  limited clinical situations in which low and transient expression of the transgene is required.

14.  Ongoing efforts to optimize the plasmid backbone via:  tissue-specific enhancers  prevent premature silencing  It increase and stabilize the levels of transgene expression obtainable with non-viral vectors.

16.  Inactivates gene involved in disease process  Antisense specific to target gene disrupts the translation of faulty gene

18.  Aqueous compartments enclose by membrane  Protect DNA from undesirable degradation during transfection  Plasmid can be covered with lipids to form micelles or liposomes  Low efficiency due to lack of ability in term of ‘ endosomal escape.

22.  Can infect more than one type of cell  New gene can be inserted into wrong position in the cell  DNA can unintentionally be inserted into patient’s reproductive system and resultant changes will pass to next generation  Transferred gene can be over expressed  Protein in excess will be harmful  Could cause an immune reaction

23.  The main purpose is:  To provide the method of transport to deliver the formulation containing the gene vector to the intended site of action.  Minimizing the contact between the gene vector and bodily fluids prior to arrival at the intended location  Reducing this contact decreases the dilution of the vector and protects the surface of the vector from non- specific interactions that are typically detrimental to its activity.

24.  The most straightforward approach to myocardial gene delivery is the direct needle injection of the vector  However, this approach has low efficiency; transduced cells are typically observed only along the needle track  But transgene expression is usually low because of the rapid removal of the vector, which is intensified by the local inflammatory reaction initiated by needle-related tissue damage

26.  Coronary artery catheterization is:  Minimally invasive and well-established procedure that allows homogenous gene delivery to each territory of the heart  The major advantages of this approach are that it is minimally invasive and relatively safe  Thus, it is especially attractive for patients with end- stage heart failure.

28.  Simplest and less invasive method  Among current available methods of cardiac gene delivery  In rodents, injection into the tail vein results in successful cardiac gene expression  Dilution by the systemic blood circulation compromises the vector concentration in the cardiac circulation, uptake by other organs such as liver, lung, and spleen before the vectors reach the heart is another issue

29.  UTMD is an immense potential target-specific gene delivery tool.  Microbubbles (MBs) of UTMD, which may consist of lipids, albumin, saccharide, biocompatible polymers and other materials are traditionally used as ultrasound contrast agents due to their physical property of reflecting ultrasound.  Microbubble as cavitation nucleus could expand and contract under the effect of ultrasound, and disrupted when the acoustic pressure reaches a much higher level.

31.  The mechanism is based on the specific response of the microbubbles upon exposure to ultrasound,namely sonoporation.  Microbubbles may oscillate when exposed to ultrasound, and then these oscillating microbubbles may rupture. So, the gene therapy vector incorporated with microbubbles can be released with high local concentrations at the site of interest.  Meanwhile, the destruction of MBs may transiently induce transient holes in membranes and cause entry of gene into target cells.

33.  Low toxicity  Low immunogenicity  Low invasiveness  MB can be intravenously injected  Great potential for repetitive application  Organs can be targeted with high specificity  Improve the efficiency  Regarded as a new choice for gene therapy

34. UTMD constitutes the most efficient method to deliver transgene up till now. Translation of gene therapies into routine medical practice will require the development and optimization of novel delivery systems capable control of gene vector biodistribution and activity.

35.  Zhi-Yi Chen1, Yan Lin1, Feng Yang1, Lan Jiang1 and Shu ping Ge2Gene therapy for cardiovascular disease mediated by ultrasound and microbubbles, Chen et al. Cardiovascular Ultrasound 2013, 11:11.  Lisa Tilemann, Kiyotake Ishikawa, Thomas Weber, and Roger J. Hajjar, Gene Therapy for Heart Failure, Circ Res. 2012 March 2; 110(5): 777–793.  Julie A. Wolfram, PhD; J. Kevin Donahue, MD, Gene Therapy to Treat Cardiovascular Disease  Elizabeth G. Nabel MD,Gene Therapy for Cardiovascular Disease,2013

36.  Ilia Fishbein, Michael Chorny, and Robert J Levy,Site-specific gene therapy for cardiovascular disease,March 2010