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

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.)