Nanobiotechnological applications in dna therapy


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Gene therapy is a form of molecular medicine that has the potential to influence significantly human health in this 21st century. It promises to provide new treatments for a large number of inherited and acquired diseases (Verma and Weitzman, 2005). The basic concept of gene therapy is simple which includes introduction of a piece of genetic material into target cells that will result in either a cure for the disease or a slowdown in the progression of the disease. To achieve this goal, gene therapy requires technologies capable of gene transfer into a wide variety of cells, tissues, and organs. A key factor in the success of gene therapy is the development of delivery systems that are capable of efficient gene transfer in a variety of tissues, without causing any associated pathogenic effects. Vectors based upon many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, currently offer the best choice for efficient gene delivery.

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Nanobiotechnological applications in dna therapy

  1. 1. Nawkar Ganesh Mahadeo
  2. 2. Key Interactions Between Fields of Biology and Nanotechnology Model Biology Nanotechnology Tools
  3. 3. DNA / Gene Therapy DNA / Gene therapy is defined as the transfer of genetic material into a cell for therapeutic benefit A "correct copy" or "wild type" gene is inserted into the genome The most common type of vectors are viruses Target cells such as the patients liver or lung cells are infected with the vector It promises to provide new treatments for a large number of inherited and acquired diseases (Verma and Weitzman, 2005)
  4. 4. DNA Vaccines DNA vaccines consist of a DNA molecule, generally a circular plasmid, with a gene that codes for the protein against which an immune response is desired The first demonstration of a plasmid-induced immune response was when mice inoculated with a plasmid expressing human growth hormone elicited antibodies instead of altering growth (Tang et al., 1992) They are capable of providing a broad, long lasting immune response They are relatively simple, cheap and quick to produce and they are stable at room temperature
  5. 5. DNA Vaccination vs Gene Therapy DNA Vaccines  Gene Therapy • The purpose of influencing the • The purpose of carrying out a immune system specific function • It aims to produce large • It aimed at achieving a long lasting, amounts of protein in a short physiologically matched expression span of time so as to generate of the gene, without activating the an immune response immune system • Does not requires a more • It requires advanced technologies targeted and finely tuned to target gene at specific site and technology its expression • Aimed at a short-term presence • Aimed at presence of the added of DNA in the animal is the genetic material over a longer desired result period of time • E.g. vaccines for HIV, herpes, • E.g. Single gene defect disorders, hepatitis and influenza cancer etc
  6. 6. Types of DNA / Gene Therapy Germ line DNA / Gene therapy • Germ cells - sperm or eggs, are modified by the introduction of functional genes • Results in heritable change • Prohibited for application in human beings Somatic cell DNA / Gene therapy • The gene is introduced only in somatic cells • Expression of the introduced gene relieves/ eliminates symptoms of the disorder • Effect is not heritable • Somatic cell therapy is the only feasible option
  7. 7. Genetic Diseases Potential Candidates for Gene TherapyDefective gene Disease1. Adenosine deaminase Severe Combined Immunodeficiency2. Cystic fibrosis transmembrane regulator Cystic fibrosis3. Factor IX Hemophilia B4. Factor VIII Hemophilia A5. Glucocerebrosidase Gaucher’s Disease6. Low-density lipoprotein receptor Familial Hypercholesterolemia7. 3-Globin Sickle Cell Anemia Three of the genetic diseases listed in table are presently the subject of gene therapy clinical trials • Adenosine Deaminase deficiency using T lymphocytes, • Familial Hypercholesterolemia using hepatocytes, and • Hemophilia using fibroblasts
  8. 8. Different Methods of Gene DeliveryViral gene transfer Non-viral gene transfer1. RNA virus vectors 1. Electroporatione.g. Oncoretroviruses, 2. Microinjection Lentiviruses, 3. Naked DNASpumaviruses 4. Particle Bombardment 5. Ultrasound2. DNA virus vectors Novel gene transfere. g. Adenoviruses,Adeno-Associated Nanoparticlesviruses, Herpesvirus e.g. Liposomes, Gold Nanoparticles, Magnetic Nanoparticles
  9. 9. Viral Vector Construction ( Verma and Weitzman, 2005)
  10. 10. First Approved Gene Therapy Procedure  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 Deoxyadenosine ADA DeoxyinosineDr. W. French Andersonwith four-year old  Causes toxicity of T lymphocytesAshanthi De Silva at U.S.National Institutes ofHealth  Lack of healthy immune system
  11. 11. Gene Therapy Strategy Isolated T lymphocytes from patient and cultured in laboratory conditions The correct copy of ADA gene was introduced into the T-cells using a retroviral vector Following transduction, the cells ware grown in culture to attained significant number of cells Gene engineered cells given back to the patient in procedure similar to a blood transfusion The amount of the ADA protein in the T-cells has risen to 25% normal
  12. 12. Why ADA Deficiency was First Target of Gene Therapy? The ADA gene had been cloned earlier The gene is of average size and can easily be inserted into a retroviral vector Bone marrow transplantation vs T cell replacement The amount of the ADA protein that needs to be produced in order to maintain a functioning immune system is only 5-10 % of normal
  13. 13. Limitations of Viral Mediated DNA delivery Toxicity and immunogenicity Restricted targeting of specific cell types Limited DNA carrying capacity e.g. for rAAV, commonly reported as 4.7kb (Flotte, 2000) Production and packaging problems Recombination and random integration into host genome High cost
  14. 14. 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 (Cavazzana et al., 2000)
  15. 15. Contd… 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 (1999) • 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 ( Raper et al., 2003)
  16. 16. Nanobiotechnology Nanobiotechnology is a rapidly advancing area of scientific and technological opportunity that applies the tools and processes of nano/ microfabrication to build devices for studying biosystems Fig. Nanobiotechnology Interdisiplinary Integration Applications of nanobiotechnology are in various fields such as predictive diagnosis, medical care, drug discovery and environment
  17. 17. What is Nanoscale? “Nano” means dwarf in Greek Nanocsale : 1 nm = 1 x 10-9 m Water Nanodevices White Tennis ballmolecule blood cell Nanopores Dendrimers Nanotubes Quantum dots Nanoshells
  18. 18. The Timeframe of Nanobiotechnology
  19. 19. Applications of Different Nanoparticles in Medicine  Liposomes • 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 andLiposomes diffusion properties  Dendrimers • 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) andDendrimers gene delivery
  20. 20. Contd…  Carbon nanotubes • These are formed of coaxial graphite sheets (<100 nm) rolled up into cylinders • It exhibit excellent strength and electrical properties and are efficient heat conductors • Due to semiconductor nature of nanotubes are used asCarbon nanotubes biosensors  Magnetic nanoparticles • These are spherical nanocrystals of 10–20 nm of size with a Fe2+ and Fe3+ core surrounded by dextran or PEG molecules • Their magnetic properties make them excellent agents to label biomolecules in bioassays, as well as MRIMagnetic contrast agentsnanoparticles • Useful in targeted gene delivery
  21. 21. Contd…  Quantum dots • These are colloidal fluorescent semiconductor nanocrystals (2–10 nm) • They are resistant to photobleaching and show exceptional resistance to photo and chemical degradation • Quantum dots excellent contrast agents for Quantum dots imaging and labels for bioassays  Gold nanoparticles • These are one type of metallic nanoparticle of size <50 nm • These are prepared with different geometries, such as nanospheres, nanoshells, nanorods orGold nanocagesnanoparticles • These are excellent labels for biosensors
  22. 22. Ideal Characteristics of NP Gene Vector System A safe and efficient NP gene vector system must fulfill the following four requirements1) Particle sizes must be in the submicron range that facilitates the penetration of the NPs through the cellular membrane2) The possibility of surface modification that permits binding of NPs with the pDNA and enhances the stability of the NP-DNA complex3) Biodegradability, so that the accumulated NPs in cells could be degraded4) High transfection efficiency
  23. 23. Hurdles in DNA DeliveryFig. DNA delivery pathways with three major barriers (A) DNA–complex formation (B) Uptake (C) Endocytosis (endosome)(D) Escape from endosome (E) Degradation (edosome)(F) Intracellular release (G) Degradation (cytosol) (H) Nuclear targeting (I) Nuclear entry and expression
  24. 24. Polyion Complex (PIC) Micelles for Plasmid DNA DeliveryFig. Polymeric micelles as intelligent nanocarriers for drug and gene delivery
  25. 25. Development of Polyion Complex (PIC) Micelle Biocompatibility of the polyplexes improved by using PEG -b- polycation copolymers which electrostatically interact with pDNA to protect DNA from enzymatic and hydrolytic degradation pDNA /PEG -b-PLL micelles intravenously injected intact pDNA observed in blood circulation after 3 hr (Harada-Shiba et al., 2002) PIC micelles stabilized by disulfide cross linking The intravenous injection of cross linked PIC micelles into mice resulted in a uniform gene expression in the liver (Miyata et al., 2005) To achieve a site-specific gene delivery, polyplex micelles might be modified with targetable ligands such as peptides and antibodies (Merdan et al., 2003)
  26. 26. A-B-C type Triblock CopolymerA) PEG SegmentB) poly[(3-morpholinopropyl) aspartamide] (PMPA) as a low pKa polycationC) PLL segment (Fukushima et al., 2005)
  27. 27. Dendritic Photosensitizer for Light-induced Gene Transfer pDNA condensation = quadruplicatedcationic peptide (CP4) + nuclearlocalization signal (NLS) Anionic DPc (Dendritic phthalocya-nine) = photosensitizer MechanismCellular uptake of the ternary complexesvia endocytosis,Dissociation of DPc from the complexesin acidic vesicles due to the protonation ofthe carboxyl groups on the dendrimerperipheryEndosomal escape of the pDNA/CP4 Fig. pDNA/CP4/DPc ternary complexescomplexes to the cytoplasm upon photoirradiation (Nishiyama et al., 2005)
  28. 28. Advantages of Nanocarriers over Viral Vector They are easy to prepare and to scale-up They are more flexible with regard to the size of the DNA being transferred e.g. DNA compacted nanoparticles can contain plasmids up to 20 kb (Fink et al., 2006) They do not elicit a specific immune response and can therefore be administered repeatedly They are better for delivering cytokine genes They show little to no toxicity in the targeted tissues, and modest immune response when high concentration (Cooper, 2007; Farjo et al., 2006) Targeted gene delivery is possible
  29. 29. Future Challenges in Nanoparticle Application It is not yet possible to predict nanoparticle biodistribution according to their physicochemical properties Once nanoparticles reach their target site, and despite their small size, they do not enter into biological systems, such as cells or organelles, easily Inside the cell, nanoparticles can remain structurally unaltered, can be modified or can be metabolized More study is required about toxic effects of nanoparticles
  30. 30. Case Study
  31. 31. Objectives To develop the preparation protocol of PBCA-CTAB NPs To study its characteristics To develop the AFP-positive hepatocellular carcinoma gene therapy using the PBCA-CTAB NP–pAFP-TK complex To study the expression of pAFP-TK in vitro
  32. 32. Materials and Methods HepG2, HeLa, and 3T3 cells (American Type Culture Collection) Escherichia coli DH5 α and pAFP-TK plasmid Enhanced green fluorescent protein plasmid N1 (pEGFP-N1) from Clonetech Herpes simplex virus thymidine kinase (HSV-TK) primers A-butyl-ester cyanoacrylic-acid (BCA) from Baiyun Limited Co. Cetyltrimethylammonium bromide (CTAB) from Sigma 3-(4,5-dimethyl-2-thiazolyl)-2,5–diphenyl-2H- tetrazolium bromide (MTT) and DNase I from Sigma Equipment used • Zetasizer 3000 and AJ-III Atomic Force Microscope (AFM)
  33. 33. AFP Photograph Contd… Amplification and purification of plasmid DNA Preparation of PBCA NPs • PBCA NPs were prepared by an emulsion polymerization method • Tween 80 was dissolved in distilled water (pH 2.8) • PBCA was added in slowly and mixed by magnetic stirring at room temperature (22 °C-25 °C) for 5 hours • Centrifuged at room temperature at 5000 rpm for 15 minutes • The suspension was filtered by polyethylene terephthalate nuclear membrane filter (diameter of pores = 0.22 µm)
  34. 34. Contd… Surface modification of PBCA-CTAB NPs 1 hr incubation • 16.6 ml of PBCA NP solution (0.625%, w/v) @ 4000 rpm for 30 min + 33.33 ml of CTAB solution (0.25%, w/v) • Precipitation washed with dd H2O and resuspended in 150 mM NaCl • Mixture lyophilized to steady state Characterization of PBCA NPs and PBCA-CTAB NPs • PBCA-CTAB NPs were uniform and that the average diameters were between 80 and 200 nm • Zeta potential of the NPs revealed a positive surface charge of +15.6 mV
  35. 35. Results  Cell viability • Cytotoxicity of PBCA NPs and PBCA-CTAB NPs to HepG2 cells and 3T3 cells was estimated by MTT assay • The toxicity of NPs would suddenly Cytotoxicity of PBCA NPs strengthen with increasing concentrationNo. Cell Type Concentration of NPs1. HepG2 100 ng/µl2. 3T3 200 ng/µl Cytotoxicity of CTAB PBCA NPs
  36. 36. DNA Loading Efficiency of PBCA-CTAB NPs The difference between the total amount of pDNA added in the NP preparation buffer and the amount of non-entrapped pDNA remaining in the aqueous suspension PBCA CTAB NP solution + pDNA in 50-µL reaction system (pH = 7) Results in different kinds of PBCA CTAB- pDNA NPs in which the ratio Fig. The change in DNA loading of PBCA-CTAB NPs to pDNA was efficiency of various NPs 1:1, 5:1, 10:1, 15:1, 20:1, 30:1, and 50:1, respectively
  37. 37. Gel Retarding Analysis and Protection Effect of NPs to pDNA PBCA-CTAB-pDNA complexes (containing 2 µg pDNA) were 1:1 5:1 10:1 15:1 30:1 50:1 10:1 incubated with 2 µl of RNase-free DNase I solution (1 µg/µl) in 50 µl of reaction buffer for 15 minutes at 37 ° C The reaction was stopped by Fig. Electrophoretic mobility adding 2 µL of RQ1 DNase stop analyses of PBCA-CTAB NP–pDNA solution complexes The integrity of the pDNA was analyzed by gel electrophoresis (1% agarose)
  38. 38. In vitro Gene Transfection Efficiency The transfection efficiency of PBCA-CTAB NPs was evaluated in HepG2 cells and 3T3 cells using the enhanced green fluorescent protein (EGFP) gene as a reporter Super- Fect Transfection Reagent was used as a positive control Fig. The expression of the EGFP gene Naked pDNA was used as negative loaded by control A) PBCA-CTAB NPs expressed in HepG2 cells B) SuperFect Transfection Reagent The results, observed by inversion expressed in HepG2 cells fluorescence microscope after C) PBCA-CTAB NPs expressed in 3T3 cells transfection D) SuperFect Transfection Reagent expressed in 3T3 cells.
  39. 39. RT-PCR Analysis To detect the expression of the HSV-TK gene Expression of β-actin mRNA was detected as an internal standard Fig. Expression of the TK gene in three PBCA NPs modified with CTAB kinds of cells transfected by different pDNAs can enter the cells effectively with exogenous genes, which *(pAFP-TK gene, p3.1-TK and pcDNA3.1) can also normally express in cells The expression of the TK gene in the AFP-positive cells was controlled byAFP enhancer more strongly than cytomegalovirus
  40. 40. Sensitivity of Transfected Cells to GCV MTT assay was used to examine the sensitivity of GCV to transfected HepG2 cells The concentration of GCV was 10 µg/ml then the cell viability was not influenced The concentration of GCV was 50 µg/ml then 50% of cells were killed Fig. Examination by MTT assay of sensitivity to GCV of transfected HepG2 cells GCV had a dose-dependent effect on survival of AFP-positive cells
  41. 41. Apoptosis Induced by PBCA-CTAB NP - Mediated pAFP - TK/ GCV System HepG2 cells transfected by pAFP- TK loaded by PBCA-CTAB NPs Stained using Hoechst 33258 stain After treatment of GCV, the nucleus of cells was condensed Fig. Apoptosis of cells induced by GCV after transfection by pAFP-TK plasmid It confirmed one of the C)Fluorescence staining of transfected mechanisms of the lethal effect of HepG2 cells not treated by GCV the PBCA-CTAB NP-mediated D)Fluorescence staining of transfected pAFP-TK/GCV system HepG2 cells treated by GCV
  42. 42. Summary Developed a novel transfection vector, PBCA-CTAB NPs - a non-viral vector that can deliver DNA into targeted cells The pAFP-TK/GCV suicide gene therapy system have a high transfection efficiency in AFP-positive cells and potent antitumoral activities in vitro The system may be an effective candidate vector for treatment of AFP-positive tumors