This document discusses targeting brain endothelial cells for gene therapy to treat juvenile neuronal ceroid lipofuscinosis (JNCL). Researchers will inject adeno-associated virus (AAV) particles containing the CLN3 gene intravenously into JNCL mice. This is intended to deliver the CLN3 gene to endothelial cells lining the brain vasculature. Restoring the CLN3 gene in these cells may help them function properly and could prevent JNCL symptoms in mice. The mice will then be tested on motor tasks to see if their condition is improved compared to untreated JNCL mice. If successful, this approach could be advanced as a potential gene therapy for human JNCL patients.

1. 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
CLN3 gene
2) Intravenous injection
For 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.
WHAT THIS MEANS FOR JNCL
THERAPY
Endothelial cells from JNCL mouse brain
have trouble internalizing molecules
CLN3 positive
endothelial
cells
outside of cell
plasma membrane
inside of cell
caveolin-1
CLN3 negative
endothelial
cells
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.
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
JNCL
Juvenile 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.
CLN3
CLN3 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
Mary Moye-Rowley, Iowa City, IA
Cross-sectional view of
a microvessel illustrates
extensive cellular
communication
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?
2
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.
3
Gene therapy to restore CLN3 to BBB
endothelial cells
1) Production of AAV-CLN3
AAV 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.