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Eradication of Solid Tumor via   Gancyclovir-based Activation ofVP22-tk Toxicity and Liposomal Toxin               Deliver...
Relevance• Prevalence and Challenges native to Breast Cancer  ▫ Second most prevalent form of cancer amongst females  ▫ Re...
Overview• Autocrine Signal Manipulation (Prolactin)• CE Pluripotency Manipulation• Engineered to contain Suicide Gene unde...
Expected Benefits• Combination of Suicide Gene/Prolactin  Trigger will allow for Selective  Cytotoxicity• Spares non-cance...
Specific Pathways of Exploitation - 1• Prolactin  ▫ Strong Association b/w High Serum Prolactin    Levels and Rapid Mammar...
Specific Pathways of Exploitation - 2• Angiogenesis Exploitation  ▫ Use of Modified Capillary Endothelial    Progenitor Ce...
Prodrug                    Toxic Compound Pathway           • Neither Enzyme in SG nor Prodrug Toxic             Individua...
Mechanisms of Destruction• 2 Effects of Toxic Product at Tumor Sites: ▫ Death of Modified CE Cells ▫ “Bystander Effect”• T...
Limitations of Current            Therapies• Percentage of modified cells which  differentiate into CE not high enough.• M...
Mechanism of Destruction - Targeted       Microcapsular Delivery• Architecture of Microcapsule ▫ Lipid Based Outer Coating...
Microcapsular Size         • Counterclockwise from           upper-left:           ▫ Engulfing of beads             under ...
Therapy Outline - I                                 Therapy Outline                         Scope and Screening       Brea...
Therapy Outline - 2             Outline - 2 (fig. 4)   Harvested CE Progenitor CellsIncorporate Synthetic Gene Construct  ...
Therapy Outline - 3Cell Culture in Neomycin - Selects cells which have been effecively modified              Culture in Ti...
Therapy Outline - 4              Microcapsule Delivery    Delivery of Toxin-Loaded Microcapsules    Cells will be Homing t...
Therapy Outline - 5         Administration of Modified Cells3-4 Day Incorporation Period into Angiogenic sites            ...
Therapy Outline - 6                     Administration of Prodrug                         Inject GanciclovirConverts to To...
Therapy Outline - 7       Activate Microcapsules via Ultrasound      Microcapsule Disruption/Drug ReleaseDeath of Tumor Re...
Emergency Extraction Plan• Toxicity Mediated Sepsis ▫ Stop administration of ganciclovir and the   ultrasound microcapsule...
Potential Drawbacks of Approach• Contingent upon Tumor Engaging in Active  Angiogenesis• Tumor Cells Halt Autocrine/Paracr...
Benefits of Proposed Approach• Specific Targeting of Tumor Regions ▫ Spares other tissues ▫ Allows for very strong agents ...
Benefits - 2• Destruction of Tumor Vasculature/Nutrient  Supply as well as Neoplastic Cells• Can destroy small, intravasat...
Benefits - 3• Ex Vivo - Transgenes delivered only to  desired cells• Transgenic Cassette maintained in dipolid  cells with...
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Cellular Engineering - Milap Thaker, Matt Lawler, W. Cartwright et. al Eradication of Solid Tumor via Gancyclovir-based Activation of VP22-tk Toxicity and Liposomal Toxin Delivery

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Eradication of Solid Tumor via Gancyclovir-based Activation of VP22-tk Toxicity and Liposomal Toxin Delivery

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Transcript of "Cellular Engineering - Milap Thaker, Matt Lawler, W. Cartwright et. al Eradication of Solid Tumor via Gancyclovir-based Activation of VP22-tk Toxicity and Liposomal Toxin Delivery"

  1. 1. Eradication of Solid Tumor via Gancyclovir-based Activation ofVP22-tk Toxicity and Liposomal Toxin Delivery Cellular Engineering - K. Parker, Professor Lawler, M. • Cartwright, W. • Thaker, M.
  2. 2. Relevance• Prevalence and Challenges native to Breast Cancer ▫ Second most prevalent form of cancer amongst females ▫ Resistance to treatment ▫ Increased Metastatic Potential/Tumor Growth due to Autocrine Signaling ▫ Autocrine Signaling Increased Angiogenesis
  3. 3. Overview• Autocrine Signal Manipulation (Prolactin)• CE Pluripotency Manipulation• Engineered to contain Suicide Gene under control of Chimeric Prolactin Receptor• Cells will also contain Toxin-Loaded Microcapsules
  4. 4. Expected Benefits• Combination of Suicide Gene/Prolactin Trigger will allow for Selective Cytotoxicity• Spares non-cancerous Angiogenic Regions• Localized Microcapsular Toxin Delivery leads to site-specific Chemotherapeutic Agent delivery
  5. 5. Specific Pathways of Exploitation - 1• Prolactin ▫ Strong Association b/w High Serum Prolactin Levels and Rapid Mammary Tumor Growth ▫ Role of Dopamine Agonists ▫ Shortcomings of Rodent Model vs. Human Model
  6. 6. Specific Pathways of Exploitation - 2• Angiogenesis Exploitation ▫ Use of Modified Capillary Endothelial Progenitor Cells ▫ Cultured to encourage differentiation into CE cells ▫ Modifications In Vitro  Suicide Gene  Conversion of Prodrug Toxic Compound
  7. 7. Prodrug Toxic Compound Pathway • Neither Enzyme in SG nor Prodrug Toxic Individually ▫ Cytotoxicity only present when cells expressing gene + prodrug Gancyclovir HSV-1 TK AnalogImage 1: http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/antibiotics/antivirals.htmlImage 2: http://www.strubi.ox.ac.uk/strubi/research/DKSgroup/vzk2.html
  8. 8. Mechanisms of Destruction• 2 Effects of Toxic Product at Tumor Sites: ▫ Death of Modified CE Cells ▫ “Bystander Effect”• Tumor Mass Decreased via: ▫ Direct Toxic Killing of Tumor Cells ▫ Nutrient Starvation Resulting from Breakdown of Tumor Vasculature
  9. 9. Limitations of Current Therapies• Percentage of modified cells which differentiate into CE not high enough.• Modified Cells = only small % of total CE cells• Does not differentiate between Cancerous/non-Cancerous Regions ▫ Cannot be used post-op/chronic sores/ulcers• Modified Cells could lodge anywhere ▫ Toxicity-induced Inflammation/Vascular Failure
  10. 10. Mechanism of Destruction - Targeted Microcapsular Delivery• Architecture of Microcapsule ▫ Lipid Based Outer Coating ▫ Core of Toxic Chemicals ▫ Small Enough to be Endocytosed by Cells• Disruption Mechanisms ▫ Heat ▫ Light ▫ Ultrasound
  11. 11. Microcapsular Size • Counterclockwise from upper-left: ▫ Engulfing of beads under 1 micron in diameter (fig 1 & 2) ▫ Chen, Geometric Control of Cell Life and Death - 10 micron beads engulfed w/normal function, no elevated apopototic activity
  12. 12. Therapy Outline - I Therapy Outline Scope and Screening Breast Cancer - must express autocrine signaling of Prolactin Ensure Presence of Prolactin via ELISA/RT-PCR for Prolactin mRNAHarvest Capillary Endothelial Progenitors from Bone Marrow/Peripheral Blood Administration of Dopamine Agonist (Cabergoline) Prolactin Release Inhibited/Circulating Prolactin Eliminated Remaining Prolactin comes from Autocrine Prolactin Producing Regions
  13. 13. Therapy Outline - 2 Outline - 2 (fig. 4) Harvested CE Progenitor CellsIncorporate Synthetic Gene Construct Suicide/VP22 Fusion Gene - Controlled by Gal4 Chimeric Prolactin Receptor Neomycin Resistance Marker Integrate cassette into Genome
  14. 14. Therapy Outline - 3Cell Culture in Neomycin - Selects cells which have been effecively modified Culture in Tissue Flasks Coated with Fibronectin FACS Sorting Fluorescence Activated Cell Signaling
  15. 15. Therapy Outline - 4 Microcapsule Delivery Delivery of Toxin-Loaded Microcapsules Cells will be Homing to Tumor RegionsProvdes Ideal Vector for Microcapsular Carriage CE Cells Actively Endocytose Liposomes Microcapsular Activation via Ultrasound
  16. 16. Therapy Outline - 5 Administration of Modified Cells3-4 Day Incorporation Period into Angiogenic sites Express Suicide Gene Begin VP-22 Mediated Export of Gene
  17. 17. Therapy Outline - 6 Administration of Prodrug Inject GanciclovirConverts to Toxic Drug only in Presence of Suicide Gene Expression
  18. 18. Therapy Outline - 7 Activate Microcapsules via Ultrasound Microcapsule Disruption/Drug ReleaseDeath of Tumor Related Vasculature/Bystander Effect
  19. 19. Emergency Extraction Plan• Toxicity Mediated Sepsis ▫ Stop administration of ganciclovir and the ultrasound microcapsule activation• Loss of Control over Modified Cells ▫ Teratoma?  Cease Administration of Dopamine Agonist  Continue Ganciclovir Administration
  20. 20. Potential Drawbacks of Approach• Contingent upon Tumor Engaging in Active Angiogenesis• Tumor Cells Halt Autocrine/Paracrine Prolactin Signaling• Side Effects of CE-Injection ▫ Proliferative Diabetic Retinopathy ▫ Pre-existing Capillary Proliferation-Related Conditions not eligible for treatment
  21. 21. Benefits of Proposed Approach• Specific Targeting of Tumor Regions ▫ Spares other tissues ▫ Allows for very strong agents with limited side effects• Microcapsules ▫ Allows use of chemotherapeutic agents that are highly effective, but difficult to administer via other means
  22. 22. Benefits - 2• Destruction of Tumor Vasculature/Nutrient Supply as well as Neoplastic Cells• Can destroy small, intravasated metastases previously undetected• Maintenance of Remission• Does not rely on delivery of transgenes to tumors in vivo
  23. 23. Benefits - 3• Ex Vivo - Transgenes delivered only to desired cells• Transgenic Cassette maintained in dipolid cells with intact DNA - decreases likelihood of transgenes being lost/altered.• No reliance on viral vector• Relies heavily on materials derived from patient (lipids, cells, etc.)
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