gene therapy 2 guo

• Non-viral Gene Delivery Systems (Transfection)


  • Safe, large gene size, but low efficacy and no nuclear transport
• Liposomes


  Synthetic cationic lipid bilayers transfer negatively charged DNA into cells, protecting DNA in vivo and entering cells by direct fusion
• Liposomes


  • Low transfer efficiency and no genome integration
  • High liposome and DNA concentration required, but cheap and easy to make
• PEGylated Liposome


  Improved half-life by adding polyethylene glycol to external layer
• Transferrin-Conjugated Liposome


  Transferrin binds transferrin receptors on human cells
• Antibody-modified liposomes


  Antibody coating increases specificity
• Sendai (HVJ) Viral Proteins


  Disrupt endosomal membrane to allow DNA to escape intact
• DNA binding proteins


  (HMG-1) targets foreign DNA into nucleus
• Nuclear Localization Sequence


  Within DNA, tags proteins for transport into nucleus
• PGA/PLGA (Poly Lactic Glycolic Acid)


  Increase transfection efficiency and navigate through biological barriers
• Electrostatic interactions


  Polymer and DNA arrange into nanocomplex that protect the DNA from degradation
• Naked DNA


  Uptake by muscle cells to express proteins, 99% degraded by extracellular nucleases (large amount required), very low gene transfer efficiency (low transient expression), not immunogenic, useful when expressed protein is amplified via signal transduction
• Biolistic Gene Gun


  Kinetic energy delivers gold/tungsten-DNA complex into exposed target (skin or in vitro, not deep tissues) that can withstand pressure, 5-10% transduction efficiency
• Skeletal myotubules


  Desirable targets because they lack receptors
• Glybera


  Treat lipoprotein lipase deficiency (prevent severe pancreatitis)
• ADA-SCID (Adenosine Deaminase Deficiency - Severe Combined Immunodeficiency)


  Rare autosomal recessive condition, multiple severe opportunistic infections from infancy - fatal in first year of life if untreated
• ADA-SCID


  Selectively toxic to lymphocytes due to accumulation of deoxyadenosine (dATP)
• ADA-SCID


  Easy to treat because it is caused by a single gene defect, edited cells have selective growth advantage in vivo, and only a small amount of ADA is necessary to correct disease
• Ex vivo retroviral vectors


  Restored immune systems in clinical trials
• In other research, adenovirus in vivo invaded unintended target causing death
• 1. Linked SCID


  Caused by deficiency in gamma subunit of IL-2 receptor (IL2RG gene)
• 1. Linked SCID


  Integration of autologous CD34+ cells w/ retroviral vector at oncogenic sites, IL2RG is oncogenic, and patients' genetic predisposition - caused patients to develop leukemia
• Cystic Fibrosis


  Mutated ABC transporter ion channel of chloride and thiocyanate found in membranes of epithelial cells - dysregulated epithelial fluid transport in lung, pancreas - impaired lung function, chronic respiratory infection, airway inflammation, (250 kbp) 6.1kbp after introns are spliced out
• Cystic Fibrosis


  Single gene defect, recessive condition, lung is accessible, progressive disease
• Cystic Fibrosis


  Needs adequate carrying capacity, cannot be detected by immune system, non-inflammatory / safe for lung inflammation, sufficient efficiency
• Cystic Fibrosis


  Cationic liposomes via aerosol
• Hemophilia B (Christmas Disease)


  X linked abnormal bleeding due to defective factor IX, 1.5 kbp, symptoms significantly reduced by expression of only 1.5-2% expression of factor IX
• Hemophilia B


  Normal - 5mg/ml, mild 250-500ng/ml, moderate 50-250ng/ml, severe <50ng/ml
• Hemophilia B


  Infusion of Factor IX is expensive and repetitive
• Self-complementary SC-AAV


  Highly efficient gene transfer for long term expression of Factor IX
• Hemophilia A


  More common X-linked Factor VIII mutation, 9kbp cDNA
• Hemophilia A


  Low protein level can prevent symptoms
• Leber's Congenital Amaurosis (LCA)


  Hereditary blindness caused by mutation in RPE65 gene, affecting retina
• Leber's Congenital Amaurosis (LCA)


  Treat with AAV
• Lysosomal Storage Disease


  Rare inherited conditions when body doesn't produce enzymes necessary to break down fats, sugars, or proteins
• Parkinson's Disease


  Gene transduction of tyrosine hydroxylase to synthesize dopamine, aim to restore dopamine synthesis in dorsal striatum, increase basal ganglia activity, neuroprotection from disease progression
• GNDF


  Lentiviral vector to overexpress glial cell neurotrophic factor protects dopamine terminals from toxins
• Ad-GDNF


  Protects dopaminergic neurons and improve dopamine-dependent behavioral function from lesions
• AAV-2


  Administered locally to transduce CNS, particularly efficient in regions affected by Parkinson's Disease
• Coronary Restenosis


  Re narrowing of coronary artery after stenting procedure (endovascular surgery)