Eradication of Solid Tumor via Gancyclovir-based Activation of
VP22-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 Analog
Image 1: http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/antibiotics/antivirals.html
Image 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
Prolactin Release Inhibited/Circulating Prolactin EliminatedRemaining Prolactin comes from Autocrine Prolactin Producing Regions
Administration of Dopamine Agonist (Cabergoline)
Harvest Capillary Endothelial Progenitors from Bone Marrow/Peripheral Blood
Ensure Presence of Prolactin via ELISA/RT-PCR for Prolactin mRNA
Scope and ScreeningBreast Cancer - must express autocrine signaling of Prolactin
Therapy Outline - 2
Outline - 2 (fig. 4)
Neomycin Resistance Marker
Integrate cassette into Genome
Chimeric Prolactin Receptor
Suicide/VP22 Fusion Gene
- Controlled by Gal4
Incorporate Synthetic Gene Construct
Harvested CE Progenitor Cells
Therapy Outline - 3
FACS Sorting
Fluorescence Activated Cell Signaling
Culture in Tissue Flasks Coated with Fibronectin
Cell Culture in Neomycin - Selects cells which have been effecively modified
Therapy Outline - 4
Microcapsule Delivery
Microcapsular Activation via Ultrasound
CE Cells Actively Endocytose Liposomes
Cells will be Homing to Tumor RegionsProvdes Ideal Vector for Microcapsular Carriage
Delivery of Toxin-Loaded Microcapsules
Therapy Outline - 5
Begin VP-22 Mediated Export of Gene
Express Suicide Gene
3-4 Day Incorporation Period into Angiogenic sites
Administration of Modified Cells
Therapy Outline - 6
Converts to Toxic Drug only in Presence of Suicide Gene Expression
Inject Ganciclovir
Administration of Prodrug
Therapy Outline - 7
Death of Tumor Related Vasculature/Bystander Effect
Microcapsule Disruption/Drug Release
Activate Microcapsules via Ultrasound
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