Gene therapy ex vivo method

Gene therapy, strategies, vectors, applications

1. Gene Therapy Ex vivo methods Akash Mahadev Iyer S-4 M.Sc Biochemistry Department of Biochemistry University of Kerala

2. Introduction Gene ; entire nucleic acid sequence that is necessary for the synthesis of a functional gene product (polypeptide or RNA ). When there is a mutation in the gene, then it will change the codon , which will change which amino acid is called for which will change the conformation of the protein which will change the function of the protein. Genetic disorders result from mutations in the genome. Protein function determines, in turn, the cell phenotype and cell function. When genes are changed in such a way that the proteins encoded by them are unable to perform their normal function, a genetic disorder may occur.

3. Gene therapy is a technique for correcting defective genes responsible for disease development. Treatment of diseases caused by single gene recessive disorders- Monogenic disorders (like cystic fibrosis, hemophilia, muscular dystrophy, sickle cell anemia etc).

4. Ashanti de Silva (4 years old) with severe combined immunodeficiency (SCID) treated in 1990 at NIH in Maryland Lacked functioning immune system because of a defect in gene called adenosine deaminase (ADA), which is involved in metabolism of dATP (nucleotide precursor used for DNA synthesis) Accumulations of dATP are toxic to T cells Normal gene cloned into vector introduced into nonpathogenic retrovirus.

5. Approaches For Correcting Faulty Genes An abnormal gene could be swapped for a normal gene through homologous recombination. The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function. The regulation (the degree to which a gene is turned on and off) of a particular gene could b altered- Anti sense therapy, RNAi and Ribozymes .

6. Steps involved; Identify the gene(s) responsible for the disorder. Make copies of the normal gene. Insert the copies into vectors. “Infect” the affected cells with the vectors. Activate the gene so that transcription and translation take place Therapeutic molecule (small regulatory RNA or RNA- Antisense oligonucleotides , aptamer , ribozymes , siRNAs ), rapidly eliminated from body This can be counteracted by- chemical modification of phosphate backbone or ribose sugars of these molecules, conjugation with specific ligands (PEG/ Cholesterol), association with a cationic lipid or polymer carriers. Persistence of the therapeutic gene

7. GENE THERAPY Germ Line Gene Therapy Somatic Cell Gene Therapy Vector type Non viral Viral Lipid based Peptide based Polymer based Naked Plasmid Electroporation Ballistic DNA Sonoporation Photoporation Hydroporation (Hydrodynamics) Magnetofection ESCs Sperm cells Egg cells Inorganic Particles Chemical Physical Nuclei Pronuclear Micro injection Nucleic acid based DNA/Cationic lipid ( Lipoplexes ), DNA/cationic polymer ( Polyplexes ) and DNA/cationic Polymer/cationic Lipid ( Lipopolyplexes ) Synthetic Natural Polyethylene imine (PEI), Dendrimers , and Polyphosphoesters . Proteins, Peptides, Polysaccharides. CaSO 4, Au, QDs, CNTs, fullerenes, supra molecular systems In-vivo Ex-vivo

8. SOMATIC CELL GENE THERAPY GERM LINE GENE THERAPY Therapeutic genes transferred into the somatic cells. Therapeutic genes transferred into the germ cells. E.g.. Introduction of genes into bone marrow cells, blood cells, skin cells etc. E.g.. Genes introduced into eggs and sperms. Will not be inherited later generations. It is heritable and passed on to later generations. At present all researches directed to correct genetic defects in somatic cells. For safety, ethical and technical reasons, it is not being attempted at present

9. Cells removed from body Transgene delivered Cells cultured Cells returned to the body Ex Vivo In Vivo Transgene delivered directly into host Strategies for Transgene Delivery

10. Ex Vivo Gene Transfer In ex vivo therapy (ex; out of, vivo; something alive), cells from person with a diseased condition are removed from the patient, transformed and cells are reintroduced into the patient Transfer of cloned genes into cells grown in culture. Transformed cells are selected, expanded by cell culture in vitro, then introduced into the patient. To avoid immune system rejection of the introduced cells, autologous cells are normally used (the cells are collected initially from the patient to be treated and grown in culture before being re-introduced into the same individual.) Only applicable to tissues that can be removed from the body, altered genetically and returned to the patient where they will engraft and survive for a long period of time (e.g. cells of the hematopoietic system, skin cells, etc.) Therapeutic genes are delivered using vectors

11. Ex- Vivo gene therapy involves transfer of genes in cultured cells which are then reintroduced into the patient Steps Isolation of cells (selected tissues e.g., Bone marrow) with genetic defect from a patient Growing the cells in a culture Introduction of the therapeutic gene to correct the defective gene Selection of the genetically corrected cells. Transplantation of the modified cells to patient As this therapy involves the use of patient own cells for culture and genetic correction, no adverse immunological reactions

12. To transfer the desired gene into a target cell, a carrier is required. Such vehicles of gene delivery are known as vectors Viral Vectors Non viral Vectors; engineered vectors- Naked DNA, particle based and chemical based Nonviral gene delivery methods Chemical and Physical approaches. The chemical methods include certain polymers and lipids, while the physical methods utilize physical properties and forces to transport genetic material into cells Vectors

13. An ideal vector needs to; Integrate the gene in the cells. Activate the gene. It should protect the transgene against degradation by nucleases in the extracellular matrix Easy and reproducible Target the right cells Production in high conc. per mL Avoid harmful side effects. Long term gene expression through insertion into the genome or stable episomal persistence Controllable gene expression Tissue specific expression Low immunogenecity .

14. Viruses as Vectors Most common type of vectors -genetically altered to carry normal human DNA. Viruses replicate by inserting their DNA into a host cell. Viruses insert their DNA into cells with high efficiency. The most commonly used viral vectors are derived from retrovirus, adenovirus, and adeno associated virus (AAV). Other viral vectors that have been less extensively used are derived from herpes simplex virus 1 (HSV-1), vaccinia virus, or baculovirus . The main drawbacks of using virus vectors are its immunogenicity and cytotoxicity .

15. dsDNA viruses -infect humans and other vertebrates. Cause benign infections of URT in humans Non-enveloped, does not integrate into the host genome Replicates as an episomal element in the nucleus Gene expression is short term and transient-repeated administration for sustained expression Adenoviral vectors have been used to transfer genes in vivo into the lung, liver, muscle, blood vessel, synovium , eye, peritoneum, brain, and tumors in animals Capsid proteins –Inflammatory response Nononcogenic (i.e., they do not cause tumors). Relatively easy to culture and Produced in large quantities. Adenoviruses

16. small non pathogenic, non env.ssDNA (4.7 kb long) virus A defective or “satellite” virus -depends on adenovirus (or some herpes viruses) Naturally replication-deficient virus, Replication is achieved via a helper virus (adenovirus or herpesvirus ) Found in cells that are infected with adenovirus. Non-inflammatory to the host. Small size -allows it to penetrate many body tissues. AAV integrates its DNA into a single site in the genome of animal cells (the AAVS1 site on chromosome 19 in humans). -therapeutic gene to be permanently integrated. Advantage - Replication-defective, nonpathogenic, and non immunogenic. Limitation- small transgene size (4681 nucleotides of single-stranded DNA) The virus can carry only a relatively short segment of DNA and they require helper viruses for activation, Possible insertional mutagenesis . Adeno -associated virus

17. Retrovirus 2 copies of a positive single-stranded RNA genome of 7 to 10kb. Enveloped virus ,deliver up to 8000 bases of ss RNA -yield permanent transduction (delivered genes will be present inside the cell for the remainder of its life). When delivering RNA into cells via retroviruses, RT of the RNA to cDNA takes place via the enzyme RT. cDNA integrates into the host genome via the enzyme integrase . Once integrated into the genome, it will be replicated when the cell replicates, -both of the daughter cells will have a copy of the virally delivered gene. -“permanent transduction”.

18. Random Integration via integrase - cDNA can be inserted in the middle of a housekeeping gene. -renders the housekeeping gene useless and would lower the amount of an essential protein, -leading to cell death If the cDNA were inserted into the middle of a tumor suppressor gene, -leads to transformation of the cell into a tumor cell. If the cDNA were inserted upstream of a proto-oncogene, it could serve to activate or mutate the gene into an oncogene , which will transform the cell.

19. Advantages Non-viral Vectors Non-viral methods Injection of Naked DNA Physical Methods to Enhance Delivery Electroporation Gene Gun Sonoporation Magnetofection Hydrodynamic delivery Chemical Methods to enhance Delivery Oligonucleotides Lipoplexes Polymersomes Polyplexes Dendrimers Inorganic Nanoparticles Cell-penetrating peptides Non-toxic No immune response

20. Non-Viral vectors It involves chemical and physical methods such as direct injection of naked plasmid DNA (particle bombardment), receptor-mediated endocytosis and gene transfer through liposomes , polymers, nanoparticles etc. Not efficient as viral vectors, Nonviral systems are attractive for the following reasons Very flexible with respect to size of DNA that has to be transported; They are cost-effective and easily allow large-scale production of plasmid DNA; They implicate less laborious safety testing as compared to recombinant vectors; There is no restriction on DNA insert length (although transfection efficiency decreases with increasing DNA length). Disadvantages Low gene transfer efficiency in vivo Transient expression due to lack of integration into the host genome; Immunostimulatory properties of plasmid DNA; In vivo toxicity due to accumulation of lipid components.

21. Liposomes and lipofection in gene therapy Liposomes -hollow microscopic spheres of phospholipids, and can be filled with DNA or other molecules during assembly. The liposomes will merge with the membranes surrounding most animal cells and the contents of liposome end up inside the cell - lipofection . Nonspecific –they tend to merge with the membranes of any cell. Because of the aqueous environment inside cells and tissues, the hydrophobic tails of the lipids will coalesce to form hollow liposomes , the interiors of which can contain oligonucleotides for cellular delivery.

22. Cationic Polymers Cationic polymers form stable polyplexes ( nanosize polymer-DNA complexes) with the desired DNA. Cationic polymer mediated gene delivery involves DNA complexation , complex mediated transversion of cell membrane to the cytoplasm, release of DNA, transfer of DNA into the nucleus PEI ( Polyethylenimine ), DEAE- dextran ( Dimethylaminoethyl-dextran ), PLL (Poly-L-lysine) , Chitosan ,PPE ( Polyphosphoesters ) Inorganic nanoparticles of metallic elements, inorganic salts, or ceramics produce complexes of size 10-100 nm.

23. The positively charged head group binds with negatively charged phosphate group in nucleic acids and form uniquely compacted structure called lipoplexes . Eg ; DOGS ( Dioctadecylamidoglycylspermine ), DC- Chol ( Cholesteryl 3 β- N-( di­methyl­amino­ethyl )­ carbamate hydrochloride), bis - guanidium - tren -cholesterol (BGTC) Cationic lipids

24. Dendrimers  is an artificially manufactured or synthesized molecule built up from branched units called monomers. Tree -like structure with defined molecular weights and entrapment properties Comprises a core, layers of branched repeat units emerging from the core, and functional end groups on the outside layer of repeat units 

25. NPs Gold NPs

26. Physical Methods to Enhance Delivery

27. Magnetofection

28. Suicide Strategy The basic concept of using prodrug -converting enzymes is to limit the action of a known cytotoxic drug to local tumor areas.  Targeted prodrug therapy includes the delivery of a gene that activates a nontoxic prodrug to a cytotoxic product by using viral vectors.  This method maximizes toxicity at site of vector delivery while minimizing toxic effects on distant cells.  The cDNA of the enzyme is delivered in to the tumor by a vector.  The corresponding nontoxic prodrug is applied and is taken up by tumor cells.  Since these cells have incorporated the cDNA in to their genome, they express the prodrug -converting enzyme.  Therefore, when the cells take up the drug, it is converted in to a cytotoxic drug that kills the tumor.   

29. Gene Therapy for Silencing of gene expression Anti sense Technology RNAi Therapeutic ribozymes Triple helix forming oligonucleotide gene therapy

30. Triple-helix forming oligonucleotide gene therapy ONs- (short) chains of (chemically modified) ribo - or deoxyribonucleotides . Their ability to bind to chromosomal DNA or mRNA through Watson–Crick and Hoogsteen base-pairing offers possibilities for highly specific intervention in gene transcription, translation, repair, and recombination for therapeutic applications Attacks DNA sequence of a mutated gene to prevent its transcription This therapy uses ssDNA that binds right into the groove between the double strands of mutated gene’s DNA. The triple helix thus produced , prevents transcription of DNA.

31. Biological fluids, like blood serum or intracellular liquids, contain highly active nucleases to destroy nucleic acids ONs composed of unmodified DNA or RNA are completely degraded within few hours, even before they reach their destination Solution; Development of modified nucleotides – higher resistance against enzymatic degradation-as they are not regarded as substrates by nucleases The 2’-position site for introduction of functional group, that enhance stability of ONs Phosphorothioates , 2’-O-methoxyethyl RNA, Phosphorodiamidate morpholino monomers, PNAs

32. Antisense technology and RNAi Silence gene expression and to turn off diseased genes. Antisense refers to short DNA or RNA sequences, which are designed to be complementary to a specific gene sequence. During transcription, the sequence of one strand is copied into single strand of mRNA – sense strand- coding strand -has the code to be read for making the protein. The opposite strand is called the antisense strand Concept of Antisense Technology - is to design an RNA molecule that will serve as complimentary base pair to mRNA you want to inhibit, thus blocking its translation into protein Concept of RNAi ; double stranded RNA- molecules are delivered into cells, where the enzyme Dicer chops them into 21-nt long siRNAs . The siRNAs then join with an enzyme complex called RISC, which guides the siRNAs to their target mRNA, where the bind by complimentary base pairing. The RISC complex degrades the si -RNA bound mRNA ,so they cannot bet translated to proteins. Silencing RNA are delivered using plasmid vector, liposomes , Lentiviral delivery, attachment of siRNAs to cholesterol or fatty acids Not possible for multigene diseases

33. Argonaute + Other Reg. proteins

34. Ribozymes Ribozymes are engineered RNA sequences containing enzyme activity capable of cleaving a specific mRNA sequence. Gene therapy can also be used to transfer a gene for a ribozyme into a defective cell. The ribozyme has two domains; The catalytic domain acts as an enzyme, and The recognition domain binds to a specific target RNA. Once bound, the enzyme domain cleaves the target RNA. Ribozymes may be engineered to recognize and destroy any target mRNA molecule. Transcription of this gene would result in production of the ribozyme RNA. The ribozyme would then bind and cleave the target mRNA.  Vector carrying a gene encoding for ribozyme is used for delivery to target cell.

35. CAR-T therapy Consists the infusion of engineered T cells that express a Chimeric Antigen Receptor  (CAR) on their cell membrane. CAR ( Chimeric Antigen Receptor) T cell and gene therapy -manipulates the patient’s own T cells (immune cells) to attack malignant cancer cells. CAR T cancer gene therapy is currently being studied as a treatment for many different cancer types and has been approved for different forms of leukemia and lymphoma. To be effective, the CAR T cells are engineered to identify and attack only those cells that have the same antigens your cancer cells have.

36. The most common procedure for CAR-T cell therapy starts with the extraction of T cells from the own patient, a process called leukapheresis . The T cells are then genetically modified to express a CAR and expanded  in vitro . Finally, they are reinfused into the patient, ready to fight the tumor.

37. CAR T cells- genetically engineered to express a receptor with 2 major functions Antigen recognition- through antibody binding T-cell activation-through phosphorylation of intracellular domains, CARs consist of three major domains: An ectodomain , - extracellular portion -includes antigen-recognition domain and a signal peptide. Transmembrane domain- supports CAR stability, and An endodomain .; intracellular endodomain -signal transduction to activate T cells during antigen recognition Binding of tumor target antigen -T-cell activation, proliferation, and target cell elimination.

38. Possible applications of Gene Therapy: Cystic fibrosis: The limitation is that airway epithelial cells are rapidly shed off. Severe combined immunodeficiency disease (SCID) Growth hormone deficiency: by implanting cultured myoblasts transfected with GH gene. Familial hypercholesterolemia: by introducing LDL receptor gene into hepatocytes .

39. Major problems with gene therapy: The Food and Drug Administration (FDA) has not yet approved any human gene therapy product for sale. Immune response. It reduces gene therapy effectiveness and makes repetitive rounds of gene therapy useless Viral vectors. Immune and inflammatory responses, also fears that viral vector may recover disease-causing ability Target. One does not have control over where the gene will be inserted into the genome Short-lived. Very hard to achieve any long-term benefits Multigene disorders. Disorders such as heart disease, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of many genes. 

40. What we have learnt..

41. References Gene Therapy of Cancer: Translational Approaches from Preclinical Studies to Clinical Implementation, edited by Edmund C. Lattime , Stanton L. Gerson Academic Press.  Therapeutic Oligonucleotides , Jens Kurreck , Royal Society of Chemistry  Basic and Applied Aspects of Biotechnology, Varsha Gupta, Springer  Non Viral Vectors in Gene Therapy- An Overview, Murali Ramamoorthy Gene therapy: advances, challenges and perspectives , Giulliana Augusta Rangel Gonçalve , July, 2017 Nonviral Vectors for Gene Therapy: Lipid- and Polymer-based Gene Transfer  Academic Press Biotechnology Fundamentals; Firdos Alam Khan, CRC press Biotechnology; Applying the Genetic Revolution, David P. Clark, Nanette J. Pazdernik , Elsevier Academic Press An Introduction to Molecular Biotechnology: Fundamentals, Michael Wink, John Wiley & Sons  Polymers and Nanomaterials for Gene Therapy ; Ravin Narain , Woodhead Publishing 

42. Thank You