This document proposes a therapy to eradicate solid tumors using genetically engineered capillary endothelial progenitor cells. The cells will be modified to contain a suicide gene regulated by a chimeric prolactin receptor and loaded with toxin-containing microcapsules. When administered to a patient, the cells will home to tumor sites where prolactin is produced, activate the suicide gene, and release toxins from ruptured microcapsules to destroy the tumor through a "bystander effect". The therapy aims to selectively target cancerous regions while sparing healthy tissue.

1. 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.

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. 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. 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. 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. 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. 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

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. 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. 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. 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. 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 mRNA
Harvest 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. Therapy Outline - 2
Outline - 2 (fig. 4)
Harvested CE Progenitor Cells
Incorporate Synthetic Gene Construct
Suicide/VP22 Fusion Gene
- Controlled by Gal4
Chimeric Prolactin Receptor
Neomycin Resistance Marker
Integrate cassette into Genome

14. Therapy Outline - 3
Cell Culture in Neomycin - Selects cells which have been effecively modified
Culture in Tissue Flasks Coated with Fibronectin
FACS Sorting
Fluorescence Activated Cell Signaling

15. Therapy Outline - 4
Microcapsule Delivery
Delivery of Toxin-Loaded Microcapsules
Cells will be Homing to Tumor Regions
Provdes Ideal Vector for Microcapsular Carriage
CE Cells Actively Endocytose Liposomes
Microcapsular Activation via Ultrasound

16. Therapy Outline - 5
Administration of Modified Cells
3-4 Day Incorporation Period into Angiogenic sites
Express Suicide Gene
Begin VP-22 Mediated Export of Gene

17. Therapy Outline - 6
Administration of Prodrug
Inject Ganciclovir
Converts to Toxic Drug only in Presence of Suicide Gene Expression

18. Therapy Outline - 7
Activate Microcapsules via Ultrasound
Microcapsule Disruption/Drug Release
Death of Tumor Related Vasculature/Bystander Effect

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. 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. 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. 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. 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.)