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Sree Prakash Pandey (NAs & Gene Therapy).pptx

The document discusses nucleic acid based therapeutic delivery systems for gene therapy. It begins with an introduction to nucleic acid therapeutics including DNA and RNA. It then discusses various mechanisms for inhibition of gene expression, such as antisense mechanisms, RNA interference, aptamer selection, and ribozymes/DNAzymes. It also discusses various physical methods for targeting nucleic acids, including gene guns, naked DNA injection, hydrodynamic delivery, electroporation, photodynamic therapy, magnetoporation, and ultrasound. The document concludes with sections on gene therapy, types of gene therapy including germline and somatic gene therapy, and methods of gene therapy delivery.

Nucleic Acid Based Therapeutic Delivery System : Gene Therapy Submitted to Dr. Vivek Dave Associate Professor Head of Department Department of Pharmacy School of Health Science Presented by Mr. Sree Prakash Pandey (CUSB2006122010) M.Pharm (Pharmaceutics) 1st year (II Sem) School of Health Science 1 Central University of South Bihar, Gaya
2 • Nucleic Acid Therapeutics • Nucleic acid, DNA & RNA • What is Gene expression & regulation ? • Different mechanism for inhibition of Gene expression • Physical targeting of nucleic acids • Gene therapy • Types of Gene Therapy • Methods of Gene Therapy • Polymers and lipids in gene delivery • Application • Conclusion • References CONTENTS
3 Nucleic Acid Therapeutics
• The process of gene delivery and expression is known as transduction. • Therapeutic NAs comprise DNA (oligonucleotides, plasmid, viruses, artificial chromosomes, and bacteria) and RNA (oligonucleotides, ribozymes, siRNA, mRNA, and viruses). • The exogenous nucleic acids can be used to modify gene expression. • Oligonucleotides as therapeutics for manipulating gene expression in living cells. 4 Nucleic Acid Therapeutics Oligonucleotides is a polynucleotide whose molecules contain a relatively small number of nucleotides.
Cont… • Synthetic oligonucleotides (ODNs) are short (<30 nucleotides) nucleic acid strands (DNA or RNA), which are chemically synthesized. • Vectors that have been developed to overcome these obstacles fall into two broad categories: nonviral and viral vectors. • The nonviral vectors consist of naked DNA delivered by injection, liposomes (cationic lipids mixed with nucleic acids), nanoparticles, and other means. • Viral vectors are derived from viruses with either RNA or DNA genomes and are represented as both integrating and nonintegrating vectors. • Non-viral methods are preferred due to their low pathogenicity. 5
Nucleic acid, DNA & RNA • Nucleotides consist of a nitrogenous base, a pentose and a phosphate. • Nucleic acids are the polymers of nucleotides (polynucleotides) held by 3' and 5' phosphate bridges. • There are two types of nucleic acids, namely deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). 6
Cont… • The pentose sugar is D-ribose in ribonucleotides of RNA while in deoxyribonucleotides (deoxynucleotides) of DNA, the sugar is 2-deoxy D-ribose. • Primarily, nucleic acids serve as repositories and transmitters of genetic information. • DNA is a polymer of deoxyribonucleotides (or simply deoxynucleotides). • lt is composed of monomeric units namely deoxyadenylate (dAMP), deoxyguanylate (dGMP), deoxy-cytidylate (dCMP) and deoxythymidylate (dTMP). 7 Reference: Satyanarayana U. et al 2006
• RNA is a polymer of ribonucleotides held together by 3',5'-phosphodiester bridges. • The three major types of RNAs with their respective cellular composition are given below 1. Messenger RNA (mRNA) : 5-10% 2. Transfer RNA (tRNA) : 10-20% 3. Ribosomal RNA (rRNA) : 50-80% 8
9 What is Gene expression & regulation ?  Gene expression is the process by which the information encoded in a gene is used to direct biosynthesis of a protein molecules that produces the structure of cell.  The process of gene expression involve two main stages- Transcription a process in which ribonucleic acid (RNA) is synthesized from DNA. Translation the biosynthesis of a protein or a polypeptide in a living cell. Gene is the basic physical and function unit of heredity which is made up of DNA & store genetic information.
Cont… • Gene regulation is the process of turning genes on and off. • It is a label for the cellular processes that control the rate and manner of gene expression. • A complex set of interactions between genes, RNA molecules, proteins (including transcription factors) and other components of the expression system determine when and where specific genes are activated and the amount of protein or RNA product produced. 10 Reference: Satyanarayana U. et al 2006
11 Ribozymes and DNAzymes Antisense mechanism Aptamer selection RNA interference Antigene mechanism The inhibition of gene expression is based on following mechanism- Different mechanism for inhibition of Gene expression
12 Antigene therapy involves blocking of transcription (by antigene oligonucleotide). 1. Antigene mechanism
Cont… • In this mechanism, the Triplex-helix forming oligonucleotides are synthetic single-stranded DNA, which hybridize to purine or pyrimidine-rich region in the major groove of double-helical DNA through Hoogsteen base pairing. If a stable triple helix is formed, it prevents unwinding of double helical DNA necessary for transcription of the targeted region or blocks the binding of transcription factor complexes. • This mechanism is not considered efficient for clinical applications even though it provides an opportunity for therapeutic interventions at a very early stage. 13
14 The antisense therapy, translation is inhibited (e.g antisense oligonucleotide). 2. Antisense mechanism
Cont… • In the antisense mechanism, the Reverse-complementary oligonucleotides, which hybridize to the mRNA strand of the targeted gene. • After hybridization, antisense oligonucleotides block expression either sterically by obstructing the ribosomes or by forming an RNA-DNA hybrid, which is a substrate for RNase H enzyme, thereby causing the cleavage of target mRNA. 15
16 RNA interference (RNAi) or post-transcriptional gene silencing is a natural process in eukaryotic cells by which double-stranded RNA targets mRNA for cleavage in sequence- specific manner. 3. RNA interference
Cont..  The mechanism of RNAi involves processing of a very long (500– 1000 nucleotides) double-stranded RNA, which is cleaved into short double- stranded RNA by the DICER enzyme.  DICER, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene.  Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. 17
18 Aptamers are nucleic acid ligands (15–40 nucleotides) isolated from combinatorial oligonucleotide libraries by in vitro selection. 4. Aptamer selection
Cont… • In solution, the oligonucleotide chain forms intramolecular interactions that fold the molecule into a complex 3D shape. • Aptamers have capability to tightly and specifically bind to the target molecules ranging from small molecules to complex multimeric structures. • The therapeutic potential of aptamers arises from the fact that many aptamers targeted against proteins are able to interfere with their biological activity. 19
• Other nucleic acid agents such as microRNA are also known and so are nucleic acids with catalytic activity: ribozymes and DNAzymes. • Ribozymes are RNA molecules containing different catalytic motifs. They hybridize (bind) to the substrate RNA through Watson–Crick base pairing and cause sequence-specific cleavage. • DNAzymes are DNA molecules containing a catalytic motif and similar to ribozyme, cleave the substrate RNA after binding to it. 20 5. microRNA : Ribozymes and DNAzymes
Different mechanisms for selective inhibition of gene expression 21 RISC:- RNA- induced silencing complex siRNA :- small interfering RNA
22 Complexatio n • The assembly of complexes between the gene carrier and the nucleic acid molecules Condensatio n • pDNA is condensed into sub-micron particles with an overall positive surface charge Binding • This allows binding to the negatively charged cell surface and promote subsequent uptake via endocytosis. Overview of the transfection pathway
23 Destabilizati on • Release into the cytosol is facilitated by destabilization of endosome membrane with pH-responsive components of the gene carrier. Transcripti on • The pDNA must be imported into the nucleus for transcription. Decondensa tion • Transcriptional activity of the transgene is favored by intranuclear disposition within the euchromatin domain, as well as efficient de- condensation from the gene carrier. Replication & Retention • Long-term expression of the transgene will require replication and nuclear retention of the pDNA as well as avoiding the transgene clearance activity of the immune response. 23
24 Schematic overview of the transfection pathway
Physical forces such as electric or magnetic field, light, hydrodynamic pressure or mechanical forces are used in some strategies for NAs delivery, such as 25 Targeting of NAs Naked DNA Injection Hydrodyn amic Delivery of NAs Electropor ation Jet Injection Ultrasound (US) Gene Gun Photochem ical Internaliza tion (PCI) PHYSICAL TARGETING OF NUCLEIC ACIDS
• A gene gun is a device used to delivery of exogeneous DNA, RNA or protein to target cell. This method is called Biolistics. • Gene gun or Particle-mediated transfection (PMT) is a useful approach of non-viral gene transfer in cancer therapy. Application • DNA vaccination and cytokine gene therapy. 26 1. GENE GUN
27 Advantages  Gene gun administration of DNA is safer than parenteral injection.  Gene gun-based DNA vaccination is much more efficient than parenteral injection, and therefore the total amount of DNA required for delivery is less.
• The main advantage of this method is its simplicity, involving direct plasmid injection, but results in low levels of gene transfer. • Several reports on direct injection to various tissue sites have been documented with one example of this method being the intra-tumoral delivery of a cytosine-deaminase suicide gene. 28 2. Naked DNA Injection
• Hydrodynamic delivery employs the force generated by a relatively rapid injection of a large volume of solution into the bloodstream. • This approach introduces naked plasmid DNA into cells in highly perfused internal organs such as liver with a remarkable efficiency. 29 3. Hydrodynamic delivery • Gene delivery efficiency in this method is a function of factors which include the anatomic structure of the organ, the injection volume, and the speed of injection.
Cont… • A large variety of substances with different molecular weights and chemical structures such as small dye molecules, proteins, oligonucleotides, small interfering RNA, and linear or circular DNA fragments applied to intracellular delivery of any water-soluble compounds, small colloidal particles, or viral particles directly into cytoplasm without Endocytosis. • This method is successful for liver transfection. 30
• The Jet injector is a device narrowed with high pressure of liquid stream that penetrates the epidermis of skin to target tumor/cancerous cell. • In the jet injection method, mechanical compression is used to move fluid, including DNA, through a small orifice rapidly, in less than a second. The result is a high pressure stream penetrating tissue without using any needle for particles. • Recent studies show that this approach can efficiently express genes in the tumors and be applied for cancer therapy using minimum naked DNA. 31 4. Jet injection method
32 USED  for gene delivery into large muscles  for gene transfer into tumors,  for cardiac gene therapy.
• In this method, short-duration electric field pulses are applied to various tissues such as muscle, skin, liver, lung, and tumor after local administration of NAs to overcome the barrier of the cell Membrane. • Injection of DNA followed by local electroporation in tumors enhances gene transfer into cells applicable in delivering of a large variety of molecules and materials such as ions, drugs, dyes, tracers, antibodies, oligonucleotides, RNA and DNA, both in vivo and in vitro. 33 5. Electroporation
• Photodynamic therapy is an emerging technique which can be applied efficiently for NA delivery to a specific light-exposed site. • Photochemical internalization (PCI), in which photosensitizing compounds localized in endocytic vesicles can be excited by the light in a specific wavelength and initiate photochemical reactions. 34 6. Photodynamic therapy OPTOPORATION
35 6. Magnetoporation Magnetoporation is to deliver the nucleic acids into the cell under the influence of magnetic field with transfecting plasmid DNA int o a variety of tumor cells.
• This approach facilitates the intra-tumoral injected DNA delivery into tumor tissues and gene transfer at cellular and tissue levels • The duration of US treatment, and the amount of plasmid DNA used are important to transfection efficiency of this method • The vehicles such as gas-filled poly(D,L-lactide- coglycolide) (PLGA) microparticles were systemically used to encapsulate plasmid DNA and US was applied to destruct the gas core and release the plasmid at the target site 36 7. Ultrasound (US)
37 GENE THERAPY
GENE THERAPY HISTORY OF GENE THERAPY • The term gene therapy was first introduced at the International Congress of Genetics. • The techniques utilized by Gregor Mendel in the 1850s, which were then developed by Ronald Fischer at the beginning of the 20th century, formed the turning points in genetics. The work of both Mendel and Fischer laid the foundations of genealogy. • The material they studied was later termed as gene by the Danish botanist Wilhelm Johannsen. • The first gene therapy trial in humans was conducted at the beginning of the 1970s, and it was observed that naturally occurring DNA and RNA tumor viruses successfully delivered new genetic information to the genomes of mammal cells.38
• Gene therapy is a novel form of molecular medicine that has the potential to influence significantly human health. • It promises to provide new treatments for the large number of inherited and acquired diseases. • Gene therapy is based on the substitution or replacement of a defective gene with a functional copy or manipulation of a gene function or expression using short nucleic acids (NAs). 39 Gene therapy is the process of inserting genes into cells to treat diseases. DEFINITION GENE THERAPY
Cont… Gene-based therapy is commonly associated with gene augmentation or gene replacement therapy, where the deficient gene product is supplied with a functional version. Gene augmentation • Gene augmentation consists on the delivery of a new protein-coding gene to treat a disease. • In gene augmentation therapy (GAT), the aim of the treatment is to enhance the amount of protein product by delivering exogenous nucleic acid molecules • The exogenous nucleic acid molecules containing instruction for the expression of the deficient protein. • GAT has been applied to cancer therapy via de novo expression of suicide genes that have the ability to induce cell death on its own expression and can range from apoptosis inducer, such as caspases to enzymes that converts prodrugs to cytotoxic compound 40
41 Gene knockdown • An experimental technique by which the expression of one or more of an organism's genes is reduced. • The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. Gene repair • Targeted gene repair is a technique used to correct a mutation at a specific site in an episome or chromosome. • It uses synthetic oligonucleotides together with the cell's inherent DNA repair system to direct single base pair changes. Cont…
42 Gene Therapy Germline Gene Therapy Somatic Gene Therapy Types of Gene Therapy
Germline Gene Therapy • In germline gene therapy (GGT), germ cells such as sperm or egg cells are modified by the introduction of functional genes into their genomeas. • Modifying a germ cell causes all the organism's cells to contain the modified gene. • The change is therefore heritable and passed on to later generations. 43 There is the introduction of DNA into germ cells is passed on to the successive generation which treat the disease.
Somatic Gene Therapy • The genetic alterations in somatic cells are not carried to the next generation. • Therefore, somatic cell gene therapy is preferred and extensively studied with an ultimate objective of correcting human disease. 44 There is the insertion of a fully functional and expressible gene into a target somatic cell to correct a genetic disease permanently.
45 Can be applied to only selected tissue, e.g, bone marrow cells Cells modification are carried outside from the patient body and then transplanted back Ex-vivo Gene therapy The direct delivery of therapeutic gene into the target cells of patient Cells modification are carried inside to the patient body and then transplanted back In-vivo Gene therapy Method of Gene Therapy
Steps of Ex-vivo Gene therapy • The following steps involve to describe the technique of ex-vivo gene therapy- 46 Transplant the modified cells to the patient. Select the genetically corrected cells (stable transformants) and grow. i.Introduce the therapeutic gene to correct gene defect. i.Grow the cells in culture. i.lsolates cell with genetic defect from a patient.
Cont… VECTORS IN GENE THERAPY • The carrier particles or molecules used to deliver genes to somatic cells are referred to as vectors. • Viruses - particularly Retroviruses, RNA is the genetic material in retroviruses. As the retrovirus enters the host cell, it synthesizes DNA from RNA (by reverse transcription). • Human Artificial Chromosome (HAC) - is a synthetic chromosome that can replicate with other chromosomes, besides encoding a human protein. • Bone marrow cells - contains totipotent embryonic stem (ES) cells. These cells are capable of dividing and differentiating into various cell types. • Ex-vivo gene therapy e.g. Sickle-cell anemia, SCID, Thalassemia, etc. 47
In-vivo Gene therapy • The direct delivery of the therapeutic gene (DNA) into the target cells of a particular tissue of a patient. • Several tissues like liver, muscles, skin, spleen, lung, brain and blood cells. • Gene delivery can be carried out by viral or non-viral vector systems. 48 Viral systems Many viral vector systems have been developed for gene delivery. These include retroviruses, adenoviruses, adeno- associated viruses and herpes simplex virus. Non-viral vector systems The viral proteins often induce inflammatory responses in the host. So, non-viral vectors proposed. Pure DNA, Lipoplexes, Human artificial chromosomes, etc.
Process of In-vivo Gene therapy 49
50 POLYMERS AND LIPIDS IN GENE DELIVERY Polyplexes  Polyplexes are formed when cationic polymers interact with DNA through electrostatic interactions.  Complexation or encapsulation of nucleic acids by the positively charged polymer reduces the charge of the nucleic acid and forms particles in the nano-size range, resulting in increased permeability.  Chitosan, poly-L-lysine and polyethylene amine (PEI) are cationic polymers widely used for gene delivery.  Polyplexes having low transfection ability or lacking specificity for a target could be made more efficient by modifying the carrier.
51 Lipoplexes  In a similar manner of polyplex, the lipids with a positive charge are able to form complexes with DNA.  Such liposomes or lipoplexes have better transfection ability than neutral liposomes.  Positively charged liposomes are able to neutralize the negative charge on DNA and nucleic acids. Due to the lipidic nature of the carrier, the transfection ability of lipoplexes are generally high. New cationic liposomes as vectors which form lipoplexes with plasmid DNA have been developed for gene delivery and have shown high transfection efficiency after systemic administration.
52 SCHEMATIC REPRESENTATION FOR DELIVERY OF LIPOPLEXES
53 Nanoplexes  Nanoparticles formed by dispersion of nucleic acids in polymers as nanospheres and encapsulation of DNA by polymers such as chitosan, albumin or collagen to form nano-capsules are categorized as nanoplexes.  The entrapment of genetic material into nanoparticles constitutes a promising approach for increasing the in vitro and in vivo transfection activity.  Calcium phosphate-DNA co-precipitates have been used for gene delivery for more than 35 years. They are nontoxic, bioresorbable, easy to produce, and able to complex with the negatively charged phosphate ions of nucleic acids by interaction through calcium ions.  The use of poly(ethylene glycol) functionalized bisphosphonate (PEG-bp) produced stabilized calcium phosphonate-DNA nanoparticles which had low toxicity and sustained ability to transfect cells.
54 1. Applications of Gene Therapy in Cancer • The three conventional modalities of treatment of cancer surgery, radiotherapy, and chemotherapy are often unsuccessful in treating cancer. • Therefore, Gene therapy is the emerging fourth modality for treatment of cancer. • Various types of cancer are now active targets for gene therapy clinical trials. Prostate Cancer Breast Cancer Lung Cancer Brain Cancer Application of Gene therapy
55 2. Applications of Gene Therapy in Cardiovascular(CV) Diseases • Gene therapy is specifically important for monogenetic CV disorders like hypertension or hypercholesterolemia. • The primary goal of gene therapy is to insert the corrected form of the mutated gene into the appropriate target cell type to restore normal function. Hypertension Hypercholesterolemia and Atherosclerosis Thrombosis Ischemia
• Gene therapy is specifically important for monogenetic CV disorders like hypertension or hypercholesterolemia. • The primary goal of gene therapy is to insert the corrected form of the mutated gene into the appropriate target cell type to restore normal function.  Hypertension  Hypercholesterolemia and Atherosclerosis  Thrombosis  Ischemia 56
57 3. Applications of Gene Therapy in Kidney Diseases Glomerulonephritis and Renal Fibrosis • Glomerulonephritis is characterized by mesangial cell proliferation, extracellular matrix accumulation, and renal fibrosis leading to renal failure. • A strategy was designed to target mesangial cell growth using appropriated AS-ODN against platelet-derived growth factor (PDGF) and transforming growth factor- β (TGF-β) genes, which are responsible for mesangial cell growth. Renal Transplantation • The immune response of a host’s body to renal graft is the major problem in kidney transplantation. • Gene therapy can provide an alternative therapy to reduce immune response in renal transplantation.
58 4. Applications of Gene Therapy in Cystic Fibrosis • Cystic fibrosis (CF) is one of the most common chronic progressive and frequently fatal autosomal recessive genetic diseases affecting the body’s exocrine glands. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, located on the long arm of chromosome 7. The normal CFTR gene encodes for formation of chloride channel protein, responsible for salt balance across the cell membrane. • In CF patients, CFTR is unable to perform the normal functions, leading to dehydration of affected organs such as the sweat glands, lungs, pancreas, and digestive system. Because lungs are the primary organs affected in CF patients, pulmonary symptoms are responsible for the majority of mortalities. • Gene therapy of CF involves the insertion of a normal copy of the CFTR gene into affected cells, which could potentially correct the biochemical abnormality of these cells and thus prevent or ameliorate disease in that organ.
59 5. Applications of Gene Therapy in Hemophilia • A deficiency or dysfunction of either FVIII or FIX leads to bleeding following vascular injury. Hemophilia is an X-linked inherited bleeding disorder characterized by severe uncontrolled hemorrhagic episodes that can even be fatal. • Hemophilia A and B resulted from a subnormal level or deficiency of essential cofactors, FVIII and FIX, respectively. • The defect in FVIII and FIX synthesis occurs due to mutation in the genes synthesizing them, respectively. • Currently, hemophilia is treated with protein replacement therapy using either plasma-derived or recombinant coagulation factors. • In a clinical trial, the nonviral method was used for the delivery of plasmid containing FVIII cDNA in patients with severe hemophilia, resulting in 1–2% expression of FVIII in six patients .
60 6. Applications of Gene Therapy in Gaucher Disease • Gaucher disease (GD) is an inherited, autosomal recessive, most common lysosomal storage disorder characterized by a decreased level of enzyme β- glucosidase (glucocerebrosidase). • Enzyme replacement therapy (ERT) is the standard treatment for GD. However, it is very expensive to administer biweekly infusions of the recombinant enzyme throughout the life of the patients. • Gene therapy could provide an alternative for GD by delivering the glucocerebrosidase (GC) gene using vectors ex vivo and in vivo. In a study, insertion of the human GC cDNA into mouse fibroblasts resulted in the appearance of human enzyme activity.
Conclusion • There are a number of human diseases, both genetic and acquired, for which gene based therapeutic approaches are gaining appraisals for genetic and acquired diseases. • Gene therapy has been proven to be an incredibly powerful tool for the prevention and cure of diseases as it aims to treat the cause rather than the symptoms of diseases. • A gene medicine is composed of an expression system with a gene of interest and a gene delivery system. 61
62 • SINKO PATRICK J. & SINGH YASHVEER; “MARTIN’S PHYSICAL PHARMACY AND PHARMACEUTICAL SCIENCES; Physical Chemical and Biopharmaceutical Principles in the Pharmaceutical Sciences”; Sixth Edition 2006; Edited by- David B. Troy; Published by- Lippincott Williams & Wilkins; Printed in China; Page No. 608 to 619. • Satyanarayana U. & Chakrapani U.; “Biochemistry”; Published by : BOOKS AND ALLIED (P) LTD.; Third Revised Edition 2006; Revised Reprint 2007; Page no. 523-530 & 625-633. • Peer Dan; “Nanotechnology for the Delivery of Therapeutic Nucleic Acids”; PAN STANFORD PUBLISHING; CRC Press 2013; Taylor & Francis Group LLC. REFERENCES
63 • Mulligan Richard C.; “The Basic Science of Gene Therapy Author(s)”; Published by- American Association for the Advancement of Science; 2016. • Verma Inder M. & Matthew D. Weitzman; GENE THERAPY: Twenty-First Century Medicine; Annu. Rev. Biochem. 2005; published: March 11, 2005. • Kumar Mukesh & Misra Ambikanandan; “Applications of Gene Therapy”; Source- Sciencedirect.com. • Patil Siddhesh D., Rhodes David G. & Burgess Diane J.; DNA-based Therapeutics and DNA Delivery Systems: A Comprehensive Review; Published: April 8, 2005. • Jafari M., Soltani M., Naahidi S., Karunaratne D.N., & Chen P.; “Nonviral Approach for Targeted Nucleic Acid Delivery”; Current Medicinal Chemistry, 2012.
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Sree Prakash Pandey (NAs & Gene Therapy).pptx

  • 1.
    Nucleic Acid BasedTherapeutic Delivery System : Gene Therapy Submitted to Dr. Vivek Dave Associate Professor Head of Department Department of Pharmacy School of Health Science Presented by Mr. Sree Prakash Pandey (CUSB2006122010) M.Pharm (Pharmaceutics) 1st year (II Sem) School of Health Science 1 Central University of South Bihar, Gaya
  • 2.
    2 • Nucleic AcidTherapeutics • Nucleic acid, DNA & RNA • What is Gene expression & regulation ? • Different mechanism for inhibition of Gene expression • Physical targeting of nucleic acids • Gene therapy • Types of Gene Therapy • Methods of Gene Therapy • Polymers and lipids in gene delivery • Application • Conclusion • References CONTENTS
  • 3.
  • 4.
    • The processof gene delivery and expression is known as transduction. • Therapeutic NAs comprise DNA (oligonucleotides, plasmid, viruses, artificial chromosomes, and bacteria) and RNA (oligonucleotides, ribozymes, siRNA, mRNA, and viruses). • The exogenous nucleic acids can be used to modify gene expression. • Oligonucleotides as therapeutics for manipulating gene expression in living cells. 4 Nucleic Acid Therapeutics Oligonucleotides is a polynucleotide whose molecules contain a relatively small number of nucleotides.
  • 5.
    Cont… • Synthetic oligonucleotides(ODNs) are short (<30 nucleotides) nucleic acid strands (DNA or RNA), which are chemically synthesized. • Vectors that have been developed to overcome these obstacles fall into two broad categories: nonviral and viral vectors. • The nonviral vectors consist of naked DNA delivered by injection, liposomes (cationic lipids mixed with nucleic acids), nanoparticles, and other means. • Viral vectors are derived from viruses with either RNA or DNA genomes and are represented as both integrating and nonintegrating vectors. • Non-viral methods are preferred due to their low pathogenicity. 5
  • 6.
    Nucleic acid, DNA& RNA • Nucleotides consist of a nitrogenous base, a pentose and a phosphate. • Nucleic acids are the polymers of nucleotides (polynucleotides) held by 3' and 5' phosphate bridges. • There are two types of nucleic acids, namely deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). 6
  • 7.
    Cont… • The pentosesugar is D-ribose in ribonucleotides of RNA while in deoxyribonucleotides (deoxynucleotides) of DNA, the sugar is 2-deoxy D-ribose. • Primarily, nucleic acids serve as repositories and transmitters of genetic information. • DNA is a polymer of deoxyribonucleotides (or simply deoxynucleotides). • lt is composed of monomeric units namely deoxyadenylate (dAMP), deoxyguanylate (dGMP), deoxy-cytidylate (dCMP) and deoxythymidylate (dTMP). 7 Reference: Satyanarayana U. et al 2006
  • 8.
    • RNA isa polymer of ribonucleotides held together by 3',5'-phosphodiester bridges. • The three major types of RNAs with their respective cellular composition are given below 1. Messenger RNA (mRNA) : 5-10% 2. Transfer RNA (tRNA) : 10-20% 3. Ribosomal RNA (rRNA) : 50-80% 8
  • 9.
    9 What is Geneexpression & regulation ?  Gene expression is the process by which the information encoded in a gene is used to direct biosynthesis of a protein molecules that produces the structure of cell.  The process of gene expression involve two main stages- Transcription a process in which ribonucleic acid (RNA) is synthesized from DNA. Translation the biosynthesis of a protein or a polypeptide in a living cell. Gene is the basic physical and function unit of heredity which is made up of DNA & store genetic information.
  • 10.
    Cont… • Gene regulationis the process of turning genes on and off. • It is a label for the cellular processes that control the rate and manner of gene expression. • A complex set of interactions between genes, RNA molecules, proteins (including transcription factors) and other components of the expression system determine when and where specific genes are activated and the amount of protein or RNA product produced. 10 Reference: Satyanarayana U. et al 2006
  • 11.
    11 Ribozymes and DNAzymes Antisensemechanism Aptamer selection RNA interference Antigene mechanism The inhibition of gene expression is based on following mechanism- Different mechanism for inhibition of Gene expression
  • 12.
    12 Antigene therapy involves blocking of transcription(by antigene oligonucleotide). 1. Antigene mechanism
  • 13.
    Cont… • In thismechanism, the Triplex-helix forming oligonucleotides are synthetic single-stranded DNA, which hybridize to purine or pyrimidine-rich region in the major groove of double-helical DNA through Hoogsteen base pairing. If a stable triple helix is formed, it prevents unwinding of double helical DNA necessary for transcription of the targeted region or blocks the binding of transcription factor complexes. • This mechanism is not considered efficient for clinical applications even though it provides an opportunity for therapeutic interventions at a very early stage. 13
  • 14.
    14 The antisense therapy, translation is inhibited(e.g antisense oligonucleotide). 2. Antisense mechanism
  • 15.
    Cont… • In theantisense mechanism, the Reverse-complementary oligonucleotides, which hybridize to the mRNA strand of the targeted gene. • After hybridization, antisense oligonucleotides block expression either sterically by obstructing the ribosomes or by forming an RNA-DNA hybrid, which is a substrate for RNase H enzyme, thereby causing the cleavage of target mRNA. 15
  • 16.
    16 RNA interference (RNAi) orpost-transcriptional gene silencing is a natural process in eukaryotic cells by which double-stranded RNA targets mRNA for cleavage in sequence- specific manner. 3. RNA interference
  • 17.
    Cont..  The mechanismof RNAi involves processing of a very long (500– 1000 nucleotides) double-stranded RNA, which is cleaved into short double- stranded RNA by the DICER enzyme.  DICER, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene.  Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. 17
  • 18.
    18 Aptamers are nucleic acidligands (15–40 nucleotides) isolated from combinatorial oligonucleotide libraries by in vitro selection. 4. Aptamer selection
  • 19.
    Cont… • In solution,the oligonucleotide chain forms intramolecular interactions that fold the molecule into a complex 3D shape. • Aptamers have capability to tightly and specifically bind to the target molecules ranging from small molecules to complex multimeric structures. • The therapeutic potential of aptamers arises from the fact that many aptamers targeted against proteins are able to interfere with their biological activity. 19
  • 20.
    • Other nucleicacid agents such as microRNA are also known and so are nucleic acids with catalytic activity: ribozymes and DNAzymes. • Ribozymes are RNA molecules containing different catalytic motifs. They hybridize (bind) to the substrate RNA through Watson–Crick base pairing and cause sequence-specific cleavage. • DNAzymes are DNA molecules containing a catalytic motif and similar to ribozyme, cleave the substrate RNA after binding to it. 20 5. microRNA : Ribozymes and DNAzymes
  • 21.
    Different mechanisms forselective inhibition of gene expression 21 RISC:- RNA- induced silencing complex siRNA :- small interfering RNA
  • 22.
    22 Complexatio n • The assemblyof complexes between the gene carrier and the nucleic acid molecules Condensatio n • pDNA is condensed into sub-micron particles with an overall positive surface charge Binding • This allows binding to the negatively charged cell surface and promote subsequent uptake via endocytosis. Overview of the transfection pathway
  • 23.
    23 Destabilizati on • Release intothe cytosol is facilitated by destabilization of endosome membrane with pH-responsive components of the gene carrier. Transcripti on • The pDNA must be imported into the nucleus for transcription. Decondensa tion • Transcriptional activity of the transgene is favored by intranuclear disposition within the euchromatin domain, as well as efficient de- condensation from the gene carrier. Replication & Retention • Long-term expression of the transgene will require replication and nuclear retention of the pDNA as well as avoiding the transgene clearance activity of the immune response. 23
  • 24.
    24 Schematic overview ofthe transfection pathway
  • 25.
    Physical forces suchas electric or magnetic field, light, hydrodynamic pressure or mechanical forces are used in some strategies for NAs delivery, such as 25 Targeting of NAs Naked DNA Injection Hydrodyn amic Delivery of NAs Electropor ation Jet Injection Ultrasound (US) Gene Gun Photochem ical Internaliza tion (PCI) PHYSICAL TARGETING OF NUCLEIC ACIDS
  • 26.
    • A genegun is a device used to delivery of exogeneous DNA, RNA or protein to target cell. This method is called Biolistics. • Gene gun or Particle-mediated transfection (PMT) is a useful approach of non-viral gene transfer in cancer therapy. Application • DNA vaccination and cytokine gene therapy. 26 1. GENE GUN
  • 27.
    27 Advantages  Gene gunadministration of DNA is safer than parenteral injection.  Gene gun-based DNA vaccination is much more efficient than parenteral injection, and therefore the total amount of DNA required for delivery is less.
  • 28.
    • The mainadvantage of this method is its simplicity, involving direct plasmid injection, but results in low levels of gene transfer. • Several reports on direct injection to various tissue sites have been documented with one example of this method being the intra-tumoral delivery of a cytosine-deaminase suicide gene. 28 2. Naked DNA Injection
  • 29.
    • Hydrodynamic deliveryemploys the force generated by a relatively rapid injection of a large volume of solution into the bloodstream. • This approach introduces naked plasmid DNA into cells in highly perfused internal organs such as liver with a remarkable efficiency. 29 3. Hydrodynamic delivery • Gene delivery efficiency in this method is a function of factors which include the anatomic structure of the organ, the injection volume, and the speed of injection.
  • 30.
    Cont… • A largevariety of substances with different molecular weights and chemical structures such as small dye molecules, proteins, oligonucleotides, small interfering RNA, and linear or circular DNA fragments applied to intracellular delivery of any water-soluble compounds, small colloidal particles, or viral particles directly into cytoplasm without Endocytosis. • This method is successful for liver transfection. 30
  • 31.
    • The Jetinjector is a device narrowed with high pressure of liquid stream that penetrates the epidermis of skin to target tumor/cancerous cell. • In the jet injection method, mechanical compression is used to move fluid, including DNA, through a small orifice rapidly, in less than a second. The result is a high pressure stream penetrating tissue without using any needle for particles. • Recent studies show that this approach can efficiently express genes in the tumors and be applied for cancer therapy using minimum naked DNA. 31 4. Jet injection method
  • 32.
    32 USED  for genedelivery into large muscles  for gene transfer into tumors,  for cardiac gene therapy.
  • 33.
    • In thismethod, short-duration electric field pulses are applied to various tissues such as muscle, skin, liver, lung, and tumor after local administration of NAs to overcome the barrier of the cell Membrane. • Injection of DNA followed by local electroporation in tumors enhances gene transfer into cells applicable in delivering of a large variety of molecules and materials such as ions, drugs, dyes, tracers, antibodies, oligonucleotides, RNA and DNA, both in vivo and in vitro. 33 5. Electroporation
  • 34.
    • Photodynamic therapyis an emerging technique which can be applied efficiently for NA delivery to a specific light-exposed site. • Photochemical internalization (PCI), in which photosensitizing compounds localized in endocytic vesicles can be excited by the light in a specific wavelength and initiate photochemical reactions. 34 6. Photodynamic therapy OPTOPORATION
  • 35.
    35 6. Magnetoporation Magnetoporation is todeliver the nucleic acids into the cell under the influence of magnetic field with transfecting plasmid DNA int o a variety of tumor cells.
  • 36.
    • This approachfacilitates the intra-tumoral injected DNA delivery into tumor tissues and gene transfer at cellular and tissue levels • The duration of US treatment, and the amount of plasmid DNA used are important to transfection efficiency of this method • The vehicles such as gas-filled poly(D,L-lactide- coglycolide) (PLGA) microparticles were systemically used to encapsulate plasmid DNA and US was applied to destruct the gas core and release the plasmid at the target site 36 7. Ultrasound (US)
  • 37.
  • 38.
    GENE THERAPY HISTORY OFGENE THERAPY • The term gene therapy was first introduced at the International Congress of Genetics. • The techniques utilized by Gregor Mendel in the 1850s, which were then developed by Ronald Fischer at the beginning of the 20th century, formed the turning points in genetics. The work of both Mendel and Fischer laid the foundations of genealogy. • The material they studied was later termed as gene by the Danish botanist Wilhelm Johannsen. • The first gene therapy trial in humans was conducted at the beginning of the 1970s, and it was observed that naturally occurring DNA and RNA tumor viruses successfully delivered new genetic information to the genomes of mammal cells.38
  • 39.
    • Gene therapyis a novel form of molecular medicine that has the potential to influence significantly human health. • It promises to provide new treatments for the large number of inherited and acquired diseases. • Gene therapy is based on the substitution or replacement of a defective gene with a functional copy or manipulation of a gene function or expression using short nucleic acids (NAs). 39 Gene therapy is the process of inserting genes into cells to treat diseases. DEFINITION GENE THERAPY
  • 40.
    Cont… Gene-based therapy iscommonly associated with gene augmentation or gene replacement therapy, where the deficient gene product is supplied with a functional version. Gene augmentation • Gene augmentation consists on the delivery of a new protein-coding gene to treat a disease. • In gene augmentation therapy (GAT), the aim of the treatment is to enhance the amount of protein product by delivering exogenous nucleic acid molecules • The exogenous nucleic acid molecules containing instruction for the expression of the deficient protein. • GAT has been applied to cancer therapy via de novo expression of suicide genes that have the ability to induce cell death on its own expression and can range from apoptosis inducer, such as caspases to enzymes that converts prodrugs to cytotoxic compound 40
  • 41.
    41 Gene knockdown • Anexperimental technique by which the expression of one or more of an organism's genes is reduced. • The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. Gene repair • Targeted gene repair is a technique used to correct a mutation at a specific site in an episome or chromosome. • It uses synthetic oligonucleotides together with the cell's inherent DNA repair system to direct single base pair changes. Cont…
  • 42.
    42 Gene Therapy Germline Gene Therapy SomaticGene Therapy Types of Gene Therapy
  • 43.
    Germline Gene Therapy •In germline gene therapy (GGT), germ cells such as sperm or egg cells are modified by the introduction of functional genes into their genomeas. • Modifying a germ cell causes all the organism's cells to contain the modified gene. • The change is therefore heritable and passed on to later generations. 43 There is the introduction of DNA into germ cells is passed on to the successive generation which treat the disease.
  • 44.
    Somatic Gene Therapy •The genetic alterations in somatic cells are not carried to the next generation. • Therefore, somatic cell gene therapy is preferred and extensively studied with an ultimate objective of correcting human disease. 44 There is the insertion of a fully functional and expressible gene into a target somatic cell to correct a genetic disease permanently.
  • 45.
    45 Can be appliedto only selected tissue, e.g, bone marrow cells Cells modification are carried outside from the patient body and then transplanted back Ex-vivo Gene therapy The direct delivery of therapeutic gene into the target cells of patient Cells modification are carried inside to the patient body and then transplanted back In-vivo Gene therapy Method of Gene Therapy
  • 46.
    Steps of Ex-vivoGene therapy • The following steps involve to describe the technique of ex-vivo gene therapy- 46 Transplant the modified cells to the patient. Select the genetically corrected cells (stable transformants) and grow. i.Introduce the therapeutic gene to correct gene defect. i.Grow the cells in culture. i.lsolates cell with genetic defect from a patient.
  • 47.
    Cont… VECTORS IN GENETHERAPY • The carrier particles or molecules used to deliver genes to somatic cells are referred to as vectors. • Viruses - particularly Retroviruses, RNA is the genetic material in retroviruses. As the retrovirus enters the host cell, it synthesizes DNA from RNA (by reverse transcription). • Human Artificial Chromosome (HAC) - is a synthetic chromosome that can replicate with other chromosomes, besides encoding a human protein. • Bone marrow cells - contains totipotent embryonic stem (ES) cells. These cells are capable of dividing and differentiating into various cell types. • Ex-vivo gene therapy e.g. Sickle-cell anemia, SCID, Thalassemia, etc. 47
  • 48.
    In-vivo Gene therapy •The direct delivery of the therapeutic gene (DNA) into the target cells of a particular tissue of a patient. • Several tissues like liver, muscles, skin, spleen, lung, brain and blood cells. • Gene delivery can be carried out by viral or non-viral vector systems. 48 Viral systems Many viral vector systems have been developed for gene delivery. These include retroviruses, adenoviruses, adeno- associated viruses and herpes simplex virus. Non-viral vector systems The viral proteins often induce inflammatory responses in the host. So, non-viral vectors proposed. Pure DNA, Lipoplexes, Human artificial chromosomes, etc.
  • 49.
    Process of In-vivoGene therapy 49
  • 50.
    50 POLYMERS AND LIPIDSIN GENE DELIVERY Polyplexes  Polyplexes are formed when cationic polymers interact with DNA through electrostatic interactions.  Complexation or encapsulation of nucleic acids by the positively charged polymer reduces the charge of the nucleic acid and forms particles in the nano-size range, resulting in increased permeability.  Chitosan, poly-L-lysine and polyethylene amine (PEI) are cationic polymers widely used for gene delivery.  Polyplexes having low transfection ability or lacking specificity for a target could be made more efficient by modifying the carrier.
  • 51.
    51 Lipoplexes  In asimilar manner of polyplex, the lipids with a positive charge are able to form complexes with DNA.  Such liposomes or lipoplexes have better transfection ability than neutral liposomes.  Positively charged liposomes are able to neutralize the negative charge on DNA and nucleic acids. Due to the lipidic nature of the carrier, the transfection ability of lipoplexes are generally high. New cationic liposomes as vectors which form lipoplexes with plasmid DNA have been developed for gene delivery and have shown high transfection efficiency after systemic administration.
  • 52.
    52 SCHEMATIC REPRESENTATION FORDELIVERY OF LIPOPLEXES
  • 53.
    53 Nanoplexes  Nanoparticles formedby dispersion of nucleic acids in polymers as nanospheres and encapsulation of DNA by polymers such as chitosan, albumin or collagen to form nano-capsules are categorized as nanoplexes.  The entrapment of genetic material into nanoparticles constitutes a promising approach for increasing the in vitro and in vivo transfection activity.  Calcium phosphate-DNA co-precipitates have been used for gene delivery for more than 35 years. They are nontoxic, bioresorbable, easy to produce, and able to complex with the negatively charged phosphate ions of nucleic acids by interaction through calcium ions.  The use of poly(ethylene glycol) functionalized bisphosphonate (PEG-bp) produced stabilized calcium phosphonate-DNA nanoparticles which had low toxicity and sustained ability to transfect cells.
  • 54.
    54 1. Applications ofGene Therapy in Cancer • The three conventional modalities of treatment of cancer surgery, radiotherapy, and chemotherapy are often unsuccessful in treating cancer. • Therefore, Gene therapy is the emerging fourth modality for treatment of cancer. • Various types of cancer are now active targets for gene therapy clinical trials. Prostate Cancer Breast Cancer Lung Cancer Brain Cancer Application of Gene therapy
  • 55.
    55 2. Applications ofGene Therapy in Cardiovascular(CV) Diseases • Gene therapy is specifically important for monogenetic CV disorders like hypertension or hypercholesterolemia. • The primary goal of gene therapy is to insert the corrected form of the mutated gene into the appropriate target cell type to restore normal function. Hypertension Hypercholesterolemia and Atherosclerosis Thrombosis Ischemia
  • 56.
    • Gene therapyis specifically important for monogenetic CV disorders like hypertension or hypercholesterolemia. • The primary goal of gene therapy is to insert the corrected form of the mutated gene into the appropriate target cell type to restore normal function.  Hypertension  Hypercholesterolemia and Atherosclerosis  Thrombosis  Ischemia 56
  • 57.
    57 3. Applications ofGene Therapy in Kidney Diseases Glomerulonephritis and Renal Fibrosis • Glomerulonephritis is characterized by mesangial cell proliferation, extracellular matrix accumulation, and renal fibrosis leading to renal failure. • A strategy was designed to target mesangial cell growth using appropriated AS-ODN against platelet-derived growth factor (PDGF) and transforming growth factor- β (TGF-β) genes, which are responsible for mesangial cell growth. Renal Transplantation • The immune response of a host’s body to renal graft is the major problem in kidney transplantation. • Gene therapy can provide an alternative therapy to reduce immune response in renal transplantation.
  • 58.
    58 4. Applications ofGene Therapy in Cystic Fibrosis • Cystic fibrosis (CF) is one of the most common chronic progressive and frequently fatal autosomal recessive genetic diseases affecting the body’s exocrine glands. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, located on the long arm of chromosome 7. The normal CFTR gene encodes for formation of chloride channel protein, responsible for salt balance across the cell membrane. • In CF patients, CFTR is unable to perform the normal functions, leading to dehydration of affected organs such as the sweat glands, lungs, pancreas, and digestive system. Because lungs are the primary organs affected in CF patients, pulmonary symptoms are responsible for the majority of mortalities. • Gene therapy of CF involves the insertion of a normal copy of the CFTR gene into affected cells, which could potentially correct the biochemical abnormality of these cells and thus prevent or ameliorate disease in that organ.
  • 59.
    59 5. Applications ofGene Therapy in Hemophilia • A deficiency or dysfunction of either FVIII or FIX leads to bleeding following vascular injury. Hemophilia is an X-linked inherited bleeding disorder characterized by severe uncontrolled hemorrhagic episodes that can even be fatal. • Hemophilia A and B resulted from a subnormal level or deficiency of essential cofactors, FVIII and FIX, respectively. • The defect in FVIII and FIX synthesis occurs due to mutation in the genes synthesizing them, respectively. • Currently, hemophilia is treated with protein replacement therapy using either plasma-derived or recombinant coagulation factors. • In a clinical trial, the nonviral method was used for the delivery of plasmid containing FVIII cDNA in patients with severe hemophilia, resulting in 1–2% expression of FVIII in six patients .
  • 60.
    60 6. Applications ofGene Therapy in Gaucher Disease • Gaucher disease (GD) is an inherited, autosomal recessive, most common lysosomal storage disorder characterized by a decreased level of enzyme β- glucosidase (glucocerebrosidase). • Enzyme replacement therapy (ERT) is the standard treatment for GD. However, it is very expensive to administer biweekly infusions of the recombinant enzyme throughout the life of the patients. • Gene therapy could provide an alternative for GD by delivering the glucocerebrosidase (GC) gene using vectors ex vivo and in vivo. In a study, insertion of the human GC cDNA into mouse fibroblasts resulted in the appearance of human enzyme activity.
  • 61.
    Conclusion • There area number of human diseases, both genetic and acquired, for which gene based therapeutic approaches are gaining appraisals for genetic and acquired diseases. • Gene therapy has been proven to be an incredibly powerful tool for the prevention and cure of diseases as it aims to treat the cause rather than the symptoms of diseases. • A gene medicine is composed of an expression system with a gene of interest and a gene delivery system. 61
  • 62.
    62 • SINKO PATRICKJ. & SINGH YASHVEER; “MARTIN’S PHYSICAL PHARMACY AND PHARMACEUTICAL SCIENCES; Physical Chemical and Biopharmaceutical Principles in the Pharmaceutical Sciences”; Sixth Edition 2006; Edited by- David B. Troy; Published by- Lippincott Williams & Wilkins; Printed in China; Page No. 608 to 619. • Satyanarayana U. & Chakrapani U.; “Biochemistry”; Published by : BOOKS AND ALLIED (P) LTD.; Third Revised Edition 2006; Revised Reprint 2007; Page no. 523-530 & 625-633. • Peer Dan; “Nanotechnology for the Delivery of Therapeutic Nucleic Acids”; PAN STANFORD PUBLISHING; CRC Press 2013; Taylor & Francis Group LLC. REFERENCES
  • 63.
    63 • Mulligan RichardC.; “The Basic Science of Gene Therapy Author(s)”; Published by- American Association for the Advancement of Science; 2016. • Verma Inder M. & Matthew D. Weitzman; GENE THERAPY: Twenty-First Century Medicine; Annu. Rev. Biochem. 2005; published: March 11, 2005. • Kumar Mukesh & Misra Ambikanandan; “Applications of Gene Therapy”; Source- Sciencedirect.com. • Patil Siddhesh D., Rhodes David G. & Burgess Diane J.; DNA-based Therapeutics and DNA Delivery Systems: A Comprehensive Review; Published: April 8, 2005. • Jafari M., Soltani M., Naahidi S., Karunaratne D.N., & Chen P.; “Nonviral Approach for Targeted Nucleic Acid Delivery”; Current Medicinal Chemistry, 2012.
  • 64.