Gene Delivery PDF

Gene Delivery Learning Objectives: 1. Explain why vectors are needed for gene delivery 2. Describe the differences between viral and non-viral gene delivery 3. Describe steps involved in developing a viral vector 4. Describe various non-viral vectors and their advantages over viral vectors 5. Ex...

Gene Delivery Learning Objectives: 1. Explain why vectors are needed for gene delivery 2. Describe the differences between viral and non-viral gene delivery 3. Describe steps involved in developing a viral vector 4. Describe various non-viral vectors and their advantages over viral vectors 5. Explain Basic concepts in physical methods of gene delivery 6. Describe the applications of gene therapy 7. Explain the barriers for oligonucleotide delivery Gene Therapy: Insertion of genes into specific somatic cells is called somatic gene therapy. They function during the life time of an individual and hence correct a genetic disease. Insertion of genes into germ cells ( fertilized eggs) is called germ line therapy. The inserted genes would be passed on to future generations also. Plasmids:  Plasmids are extra chromosomal genetic elements which are circular in shape with double-stranded DNA molecule with the capacity for autonomous replication in cells.  In general, the chromosome carries all the genes essential for growth, whereas plasmids carry optional genes that confer additional properties. Hence cells that have lost plasmids are still viable. Why are vectors essential for gene delivery?  Can’t enter the cell on its own  DNA is degraded in-vivo by Endo nucleases.  Degraded in the cytoplasm and can’t enter the Nucleus Vectors in gene therapy Virus vector Non-viral vector Advantage: Advantages: safe, transfer high protein large amount of transgene production Disadvantages: low level of Disadvantages: protein production and immunogenicity, transient expression mutation, size limitation of transgene Retroviruses:  Retroviruses are RNA viruses that have the ability to insert their genes permanently into host cell chromosomes after infection.  For gene expression, retrovirus must reverse transcribe its RNA genome into double-stranded DNA, which is then integrated into host cell DNA.  Generally, retroviruses infect only dividing cells and thus are often used to introduce genes into cells ex-vivo where cell division can be stimulated with growth promoting media.  Retroviral vectors can also be directly administered to patients, though the applicability of this approach is limited by the rapid inactivation of the retroviruses by human complement.  Retroviral vectors are not safe to use because of its random insertion into host cell chromosome, which may lead to insertional mutagenesis and oncogenesis. Viral genome components: gag: codes for core proteins pol: codes for reverse transcriptase env: codes for viral protein coat LTRs (long term repeat sequences): which include promoter/enhancer regions and sequences involved with integration Ψ or Ø : packaging sequence Packaging cell line: It is a recombinant cell line having essential sequences for viral replication and packaging. e.g.: HEK 293 cells, Hela-E1 cells  Retroviral construction for gene delivery Gavin Brooks, Gene therapy, 1st ed., 2002, Pharmaceutical Press, London.  med endosome Patientcell celldivision randomly inserted intopatientsgenome Retroviral mediated gene delivery Gavin Brooks, Gene therapy, 1st ed., 2002, Pharmaceutical Press, London.  Adenovirus Receptor mediated cell entry: αv integrins, CAR (coxsackie-adenovirus) [figure from Levine, Viruses, Scientific American Library, 1992] Adenoviral vector construction Gavin Brooks, Gene therapy, 1st ed., 2002, Pharmaceutical Press, London. Adenoviral-mediated gene delivery Gavin Brooks, Gene therapy, 1st ed., 2002, Pharmaceutical Press.  Gene Therapy-Success stories 1990: Severe combined immunodeficiency (SCID) – Ashanti DeSilva was treated for SCID – Patient’s WBC were collected and inserted the missing gene then put them back into her blood stream. – This strengthened her immune system 2006: NIH scientists successfully treated metastatic melanoma in two patients 2011: Gero Hutter treated a man with HIV using gene therapy  Risk with viral vectors 1999: Patient suffered from a partial deficiency of ornithine transcarbamylase (OTC)- ammonia accumulates in blood. Treated with adenovirus vector to deliver OTC gene to the liver 4 hours after treatment, the patient developed a high fever, within four days of treatment, patient died from multiorgan failure (systemic delivery of the vector triggered a massive inflammatory response that led to disseminated intravascular coagulation, acute respiratory distress and multiorgan failure 2002–2003: Retrovirus vector induces a lymphoproliferative disorder Three young children suffering from the fatal SCID syndrome had developed functional immune systems after the reinfusion of haematopoietic stem cells that were transduced ex vivo with a retroviral vector that carried the gene encoding the missing cytokine receptor. After a few years patients developed a leukemia-like disorder. Non-Viral vectors Components of an ideal non-viral vector: 1. Module to interact with DNA 2. Targeting moiety 3. Endosomolytic agent 4. Nuclear localization sequence  Non-viral vectors Calcium Phosphate Precipitation Precipitation of DNA – Mix DNA with calcium chloride – Add in a controlled manner to a buffered PBS solution Disperse the precipitate onto the cultured cells Uptake by the cells via endocytosis or phagocytosis Calcium phosphate protects against intracellular and serum nucleases Inexpensive, safe, low toxicity Transfection efficacy below 10% (in vitro) Transfection either transient or stable Ineffective in vivo Cationic Lipids One of the best non-viral vectors Cationic lipids assemble and entrap plasmid DNA Dioleoyltrimethylammo – Cationic headgroup to condense DNA nium propane (DOTAP) – Lipid moiety for fusogenic cellular entry – DNA/cationic lipids form multilamellar bilayer complexes Uptake via endocytosis Off-the-shelf transfection agent – Mix plasmid and lipids for 30 minutes and place over cells Downside: – Toxic at higher concentration Thomas et al., Nat. Rev. Genetics, 2003, 4, 346 Pack et al., Nat. Rev. Drug Discov. 2005, 4, 581  Cationic polymers Contain positive charged groups formation of polyplexes with DNA Relatively inexpensive Ability to incorporate ligands Parameters that influence transfection and toxicity – Charge ratio – Distance of charge from backbone – Type of charge – Complex aggregation Thomas et al., Nat. Rev. Genetics, 2003, 4, 346 Pack et al., Nat. Rev. Drug Discov. 2005, 4, 581  Cationic polymers: Polyplexes are internalized by endocytosis and polyplexes are released from endosomes by ‘Proton sponge’ effect Fate of Plasmid in vivo: Size of the plasmids Tumor vasculature has high interstitial pressure but the leaky vasculature & absence of lymphatic system results in reduced clearance (EPR effect). Plasmids by i.v. route are taken up by lungs and liver preferentially  Viral vectors Non-viral vectors Cell Entry: Adsorption (+ charge) Receptor binding proteins Bind to Heparan sulphate e.g: Integrins, CAR Proteoglycons (HSPG) Endocytosis Escape: Fusogenic proteins Fusogenic lipids Acid –sensitive liposomes Proton-sponge effect Nucleus Entry - Nuclear localization signals (e.g.: SV 40) - Active transport - During mitosis Anti sense Oligonucleotide Delivery: “Antisense oligonucleotides” are short synthetic strands of nucleic acid which bind to DNA, mRNA or extra cellular proteins in a complimentary fashion and arrest protein synthesis by inhibiting transcription and translation. Three basic approaches have been explored in this area: 1. Antisense oligonucleotides that bind to mRNA and block translation. 2. Triple- helix-forming oligonucleotides which bind in a sequence specific manner in the major groove of duplex DNA. 3. Oligonucleotides which bind to extracellular proteins and inhibit their enzymatic activity. Antisense oligonucleotide therapy Barriers for Efficient ON uptake: 1. The lipophilic cell membrane 2. ON should pass through the endosomal membrane or risk the lysosomal degradation 3. Though evidence suggests that once inside the cytosol, ONs are rapidly transported to the nucleus, nucleases within the intracellular milieu degrade ON 4. Protein binding results in less availability for uptake Kynamro® (Mipomersen sodium) FDA approved in 2013 Inhibitor of apolipoprotein B-100 synthesis indicated as an adjunct to lipid-lowering medications and diet to reduce low density lipoprotein-cholesterol (LDL-C), apolipoprotein B (apo B), total cholesterol (TC), and non-high density lipoprotein- cholesterol (non HDL-C) in patients with homozygous familial hypercholesterolemia KYNAMRO is available only through a restricted prescribing and distribution program called KYNAMRO REMS. 200 mg weekly by S.C. Vitravene ® (Fomivirsen) Antisense phosphorothioate oligonucleotide Inhibits replication of human cytomegalovitrus- used for local treatment of CMV retinitis 300 micrograms- intravitreal injection FDA Approved Oligonucleotide drugs Summary/Study points 1. What is a plasmid? Why are vectors needed for gene delivery? 2. Viral vectors vs non-viral vectors 3. Retroviral vectors -Pros and Cons 4. Adenoviral vectors-Pros and Cons 5. What are lipoplexes? Formulation optimization factors 6. What are polyplexes? Formulation optimization factors 7. What are antisense oligonucleotides ? What is their mechanism of action 8. Comparison between plasmid delivery vs ODN delivery 9. Commercial ODN products.  References 1. Vyas SP, Khar RK. Targeted and Controlled Drug Delivery, 1st ed., 2002, CBS Publishers and Distributors, New Delhi. 2. Gavin Brooks, Gene therapy, 1st ed., 2002, Pharmaceutical Press, London, UK. 3. Thomas et al., Nat. Rev. Genetics, 2003, 4, 346. 4. Pack et al., Nat. Rev. Drug Discov. 2005, 4, 581