Molecules VI. Gene Transfer and Gene Silencing

Transcription

1 Molecules VI Gene Transfer and Gene Silencing

2 The Principle of Gene Therapy Gene therapy is the introduction of genetic material into cells for therapeutic effect. Implant Construct Remove Tissue Purify / Select Target Cells Transfer Genes to Cells Create Tissue Engineered Construct

3 Why Gene Therapy? Use the body s own cells as the production facility for making therapeutic protein In principle, gene therapy can be more efficient, less expensive, & longer-lasting Therapeutic Applications

4 Applications and Targets for Gene Therapy Disease SCID Cystic fibrosis Hemophilia B Muscular dystrophy Cancer Gene Adenosine deaminase (ADA), γc receptor, others CFTR Factor IX Dystrophin/sarcoglycan p53 and others

5 p53 and Apoptosis Vogelstein and Kinzler, Nature Med., 10:789 (2004)

6 Overview of Gene-based Therapies 1972: Conceptual introduction & ethical questions T. Friedmann, Gene therapy for human disease?, Science 1990: First clinical trials (ADA deficiency) 1994: first clinical trial using antisense oligonucleotides (ONs) to block genes (HIV) 1998: FDA approval of Vitravene antisense ON for CMV retinitis 2002/03: Successes/failures of French SCID trials 2003: China FDA approves p53 gene therapy for head and neck squamous cell carcinoma 2004: first clinical trials based on sirna Present: industry invests ~ $1 billion annually in gene therapy research; NIH supports > $200 MM Over 1000 clinical trials have been initiated, but only a few have reached Phase III

7 Few Clinical Trials Reach Phase III

8 Delivery Barriers Physiological barriers Rapid clearance Immune recognition Uptake by non-target tissues Biological instability Cellular barriers Charge and steric barriers to entry Endosomal escape Cytoplasmic stability Nuclear membrane targeting & stealth moieties membrane receptors DNAliposome complex 1) BINDING OF LIGAND AND RECEPTOR 2) ENDOSOMAL UPTAKE NUCLEUS 3a) ENDOSOMAL ESCAPE & NUCLEAR TRANSLOCATION 3b) LYSOSOMAL DEGRADATION LYSOSOME Based on Int. J. Pharm., 229:1, 2001

9 Viruses are the Most Efficient Retrovirus Virus Receptor Transport RNA Reverse Reverse Transcription Lipid Bilayer Envelope protein (gp70) Binding to Receptor Fusion / Entry Entry and Uncoating DNA Protease Reverse Transcriptase Target Cell Nucleus Gene Expression LTR GENE LTR Integration Nuclear Entry and Integration Nucleus Gene Expression Capsid Nucleocapsid RNA Integrase but not the most effective Safety Titer (hard to process)

10 Vector Varieties Early 90s: mostly retrovirus Late 90s: mostly adenovirus

11 Challenges in creating artificial viruses Advanced design generates unique challenges Self-assembly Maintenance of activity Immune system avoidance Cationic polymer DNA Fusogenic peptides Nuclear targeting protein + + MULTIPLEX VECTOR

12 A New Generation of Vectors A successful delivery vector should: Entrap and condense DNA Protect DNA from nucleases Adsorb to cell surfaces Penetrate cellular membranes Escape intracellular trafficking barriers Release DNA within cell nuclei (unpackaging) Target the cells of interest selectively Be non-toxic

13 Available Materials Cationic polymers Natural materials (e.g., chitosan) Pseudo-natural materials (e.g., polylysine, lipids) Novel, synthetic materials Co-polymer blends (e.g, stabilization with PEG) Peptides (membrane penetration, nuclear targeting) Biological ligands (targeting of specific cell types)

14 Ligand Targeting The distribution of vectors can be influenced by the attachment of ligands that are recognized by receptors unique to the target cell type Limited by receptor number and trafficking routes and rates Binding agent DNA ligand Examples: receptor Ligand Receptor Cell type targeted ASGP/galactose ASGPr Hepatocytes EGF/TGF EGFR Multiple carcinoma Folate Folate R. Ovarian carcinoma MAb E-selectin Endothelial (inflammation) HPV capsid HPVR Cervical epithelial carcinoma

15 PRINCIPLE OF ANTISENSE INHIBITION BY COMPLEMENTARY BASE PAIRING 5 -A-G-G-U-C-A-C-U-U-U-G-C-A-A-C-G- 3 target mrna 3 -C-A-G-T-G-A-A-A-C-G-T-T-5 antisense DNA NORMAL PROTEIN PRODUCTION DNA DNA ANTISENSE INHIBITION mrna PROTEIN mrna NO PROTEIN antisense odn

16 ANTISENSE IN THE CLINIC SELECTED ONGOING U.S. TRIALS Compound Type of ODN Lead Indication Gene Target Phase ISIS 2922/Vitravene PS CMV Retinitis antiviral Marketed ISIS 2302/Alicaforsen PS Ulcerative colitis ICAM-1 III ISIS MOE (2 ) Type 2 diabetes PTP-1B II ISIS MOE (2 ) High cholesterol apob-100 II OGX-011 (ISIS/OncoGenex) 2 -MOE (2 ) Prostate cancer Clusterin II G3139/Oblimersen PS CLL (leukemia) bcl-2 III (Genta) Malignant melanoma I LErafAON (NeoPharm) end PS (liposomal) Neoplasms c-raf I/II HYB2055/IMOxine (Idera) Mixed backbone, CpG Renal cell carcinoma TLR9 agonist II GTI-2040 (Lorus) PS Various cancers R2 of RNR II GTI-2501 (Lorus) PS Prostate cancer R1 of RNR II AVI-4126/Resten-MP morpholino (3 ) Restenosis c-myc II/III AVI-4557 morpholino (3 ) drug metabolism CYP450 3A4 I/II CpG 7909 (Coley/Pfizer) PS, CpG NSCLC and vaccine TLR9 agonist III

17 Delivery of Antisense Oligonucleotides Common features of oligonucleotides vs. plasmid DNA with regard to delivery Nucleic acids, negatively charged Susceptible to biochemical attack No evolved mechanisms for import Unique features Size (15-25 nt vs kb) Single-stranded More susceptible to nucleases Backbone chemistry variable Cellular target is different How do these factors affect design and efficacy of antisense delivery vectors?

18 Delivery of Antisense Oligonucleotides Common features of oligonucleotides vs. plasmid DNA with regard to delivery Nucleic acids, negatively charged Susceptible to biochemical attack No evolved mechanisms for import Unique features Size (15-25 nt vs kb) Single-stranded More susceptible to nucleases Backbone chemistry variable Cellular target is different How do these factors affect design and efficacy of antisense delivery vectors?

19 CELLULAR BARRIERS TO ACTIVITY Charge repulsion EXTRACELLULAR mrna + CYTOPLASM ENDOSOME RNase H + RNase H LYSOSOME Lysosomal degradation Nuclease degradation Non-target hybridization

20 ANTISENSE ACTIVITY vs. ODN DELIVERY PEI/PO odn PEI/PS odn * Antisense levels correlate directly with levels of intracellular ODNs Sundaram et al., submitted (2007)

21 KINETIC MODELING OF DELIVERY ODNs (A) k d e A e k 1 k d i A i where PEI/PS odn Lumped parameter from model ~ Intracellular release rate PEI MW ODN chemistry PO PS 10K K K K Sundaram et al., submitted (2007)

22 mrna (M) KINETIC MODELING OF ANTISENSE ACTIVITY A i Φ M λ K H Protein (P) M ψ δ P PEI/PS odn To obtain solution, solve simultaneously mrna : Protein : where * Model predicts delayed onset & sustained activity of PS ODNs Sundaram et al., submitted (2007)

23 MECHANISM OF RNA INTERFERENCE CELL dsrna Dicer sirna RNA interference (RNAi) RISC Short interfering RNAs (sirnas) seem more potent than antisense oligonucleotides Clinical trials have recently begun (Phase I, AMD) RISC* target mrna Target sequence selection is an issue RNAi specificity and dynamics are not well understood dsrnase degraded RNA

24 Further Reading Lee and Roth, Curr. Opin. Biotechnol., 2003; 14: