Targeting Brain Endothelial cells for JNCL therapy

Colleen S Stein1, Mark Schultz2, Luis Tecedor3, and Beverly L Davidson3, 1Dept. of Internal Medicine, Carver College of Medicine, University of Iowa; 2Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI: 3Children's Hospital of Philadelphia, Philadelphia, PA. contact e-mail: colleen-stein@uiowa.edu

CLN3BDSRA 2014

WHAT THIS MEANS FOR JNCL THERAPY

CLN3 positiveendothelial

cells

Endothelial cells from JNCL mouse brain have trouble internalizing molecules

TWO OF THREE TYPES OF ENDOCYTOSIS ARE DEFECTIVE IN JNCL MOUSE BRAIN ENDOTHELIAL CELLS. Cells internalize extracellular molecules by a process called endocytosis, in which a portion of the outer membrane of the cell buds into the cell and pinches off, bringing in a "mouthful" of molecules. Three main forms of endocytosis are depicted in the drawing: clathrin-dependent, caveolar, and fluid-phase endocytosis. Red-tagged transferrin, green-tagged albumin, and green-tagged dextran are used to examine these forms of endocytosis, respectively. In this experiment we prepared cultures of endothelial cells harvested from unaffected and JNCL mouse brains and examined internalization of the red- or green-tagged molecules. Interestingly, we found that both caveolar and fluid-phase endocytic pathways are impaired in the JNCL endothelial cells, while clathrin-dependent endocytosis is intact.

inside of cell

outside of cell

plasma membrane

caveolin-1

CLN3 negativeendothelial

cells

Acknowledgements

• These studies are funded by the BDSRA, Beyond Batten Disease Foundation (BBDF), and the NIH.

• We thank the Gene Transfer Vector Core and the Central Microscopy Research Facility at the University of Iowa.

1

Art by Mark L Schultz

INTRODUCTION

JNCLJuvenile Batten disease (JNCL) is caused by mutations in the CLN3 gene, creating a deficiency or defect in the CLN3 protein. CLN3 deficiency leads to JNCL symptoms, which include loss of vision, seizures, and progressive decline in motor and mental capabilities.

CLN3CLN3 is situated within lipid membrane regions inside cells, and is not secreted. We don't know the exact function of CLN3, but it appears to play a basic role in the normal transport of proteins and lipids inside cells.

While CLN3 is found at some level in all cells throughout the body, JNCL is a disease of central nervous system. So it seems that CLN3 has a critical role in cells that control the function of the brain and eye. One important type of cell that is often overlooked in studies of neurological diseases is the endothelial cell. Endothelial cells form the inside layer of blood vessels.

The Blood-Brain Barrier (BBB)In the blood vessels (vasculature) of the brain, endothelial cells are specialized to protect and feed the brain. Here endothelial cells butt up against eachother tightly, eliminating spaces between cells. This creates a physical barrier between the blood and the brain, aptly called the blood-brain barrier (BBB). The BBB protects the brain from entry of microbes, toxic substances, and destructive immune cells. Nutrients are selectively taken into and transported through the endothelial cells. Thus the underlying brain cells (neurons and glial cells), rely on endothelial cells for nutrient supply for optimal neural performance and viability.

In our previous work, our CLN3 reporter mouse indicated that CLN3 is made at relatively high level in endothelial cells throughout the brain vasculature. Thus it is possible that CLN3 is critical to BBB functions, which dictates brain health.

Dense vasculature in the brain:Endothelial cells that line the blood vessels regulate nutrient

supply from the blood to the brain.

Astrocyte extends processes onto micro-vessels

Neurons

Cross-sectional view of a microvessel illustrates extensive cellular communication

Mary Moye-Rowley, Iowa City, IA

Adapted from Abbott et al., Nature Reviews, Neuroscience, 2006

The brain is the most densely vascularized organ in the body. This density is necessary to meet the oxygen and nutrient demands of intense neuronal activity. Every neuron and glial cell in the brain contacts or is in close proximity to a blood vessel. The endothelial cells that line the blood vessels in the brain form tight cell-to-cell junctions with each other, creating a physical barrier between the blood and the brain, termed the blood-brain barrier (BBB). Most nutrients gain entry into the brain by passing though the endothelial cells. Endothelial cells at the BBB are equipped with numerous receptors and channels, and act as gatekeepers, regulating selective transport into and out of the brain. Thus neuronal health is dependent upon endothelial cell health.

We propose that endothelial cell dysfunction is a critical event leading to JNCL symptoms, and hypothesize that restoring CLN3 to endothelial cells will protect against JNCL symptoms.

Does restoration of CLN3 to endothelial cells prevent JNCL?

USE OF GENTICALLY ENGINEERED MICE TO TEST THE HYPOTHESIS. It is conceivable that vascular endothelial cell impairment is a primary event in JNCL pathogenesis. Thus, we may be able to avert disease by providing normal CLN3 exclusively to brain endothelial cells. To test this concept, we are using transgenic mice. A transgenic mouse strain is one that has been genetically altered to carry an extra gene, referred to as a transgene.

For this project we are using JNCL mice that have been genetically engineered to carry an inducible CLN3 transgene in their genome. By breeding this mouse to a Tie2-Cre inducer mouse, we will trigger CLN3 expression in the endothelial cells of progeny mice. Progeny mice will be tested in motor and behavioral assays to assess whether they develop JNCL symptoms, or remain symptom-free or show milder disease.

2

3Gene therapy to restore CLN3 to BBB

endothelial cells

1) Production of AAV-CLN3AAV particles are microscopic virus-like particles used to delivery genes into cells. For gene therapy, we are designing AAV particles that will effectively attach to and deliver genes to brain endothelium. We will package the CLN3 gene into these AAV particles.

AAV-CLN3 GENE TRANSFER TO BBB ENDOTHELIAL CELLS. Apart from the transgenic mouse approach, another way to determine whether providing CLN3 to endothelial cells will protect against JNCL symptoms is to do Gene Therapy. Here we plan to utilize AAV virus-like-particles to transfer the CLN3 gene into endothelial cells of JNCL mice.

These studies will tell us whether blood vessel endothelium is a worthwhile target for CLN3 gene transfer in JNCL patients:

If we find that providing CLN3 to endothelial cells of the brain vasculature in the JNCL mouse, by either genetic manipulation (Fig. 2) or by gene transfer (Fig. 3) results in milder disease, this would support advancement of similar gene transfer strategies for clinical application to JNCL patients

This gene transfer approach would involve i.v. injection to achieve widespread gene transfer throughout the brain vasculature, and would not require direct injection into the brain.

CLN3 gene

2) Intravenous injectionFor gene therapy, AAV-CLN3 particles will be injected intravenously into JNCL mice. AAV-CLN3 particles will bind and deliver the CLN3 gene to endothelial cells lining the blood vessels of the brain. With a good copy of the CLN3 gene, the endothelial cells can then make CLN3 protein.

3) Therapeutic effect?JNCL mice injected with AAV-CLN3 will be monitored to determine if they exhibit superior performance on motor tasks and reduced brain pathology compared to untreated JNCL mice.

Mice will be tested for ability to maintain balance on a rotating rod. Image adapted from Nature Neuroscience 16: 658, 2013.