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"
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.
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
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
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
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
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
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
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
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
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
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
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
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
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
Therapy Outline - 5 Administration of Modified Cells3-4 Day Incorporation Period into Angiogenic sites Express Suicide Gene Begin VP-22 Mediated Export of Gene
Therapy Outline - 6 Administration of Prodrug Inject GanciclovirConverts to Toxic Drug only in Presence of Suicide Gene Expression
Therapy Outline - 7 Activate Microcapsules via Ultrasound Microcapsule Disruption/Drug ReleaseDeath of Tumor Related Vasculature/Bystander Effect
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
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
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
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
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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