Cellular Therapies December 9, 2013

Scott C. Baraban, Ph.D.

University of California, San Francisco

American Epilepsy Society | Annual Meeting

Disclosure

Neurona Therapeutics – Co-founder

BioCrea - Consultant

Dravet Syndrome Foundation – SAB

Grant Support

NIH (NINDS), C.U.R.E., DSF, AES, CIRM

American Epilepsy Society | 2013 Annual Meeting

Learning Objectives

• Understand current cell therapy approaches to control intractable seizures

• Discuss potential limitations in moving these approaches toward the clinic

American Epilepsy Society | 2013 Annual Meeting

Problem: epilepsy is a network phenomena

• increased neuronal bursting and synchronization are a hallmark of seizures

Morgan RJ, Soltesz I. Proc Natl Acad Sci 2008 Worrell G A et al. Brain 2004

Inhibition may be the best way to constrain an epileptic network

• Evidence of an inhibitory restraint of seizure activity in humans. Schevon et al. Nat Comm 2012

• How inhibition influences seizure propagation. Trevelyan AJ, Schevon CA. Neuropharmacology 2013

How do we increase inhibition?

• GABA mimetic drugs

• Modulation of postsynaptic GABA receptors

• Block GABA re-uptake

• New GABA interneurons?

A strategy for cell therapy

1. Identify epileptic zone

2. Cell transplantation

3. Cell integration and seizure control

Adapted from Parent & Murphy

News & Views; Nature Neuroscience 2013

Fine A, Meldrum BS. Neuropsychologia 1990

Modulation of experimentally induced epilepsy by intracerebral grafts of fetal GABAergic neurons

Nolte MW, Löscher W, Herden C, Freed WJ, Gernert M. Neurobiol Dis. 2008

Benefits and risks of intranigral transplantation of GABA-producing cells subsequent to the establishment of kindling-

induced seizures

Why earlier approaches did not work?

• Failure of transplanted cells to integrate

• Mixed populations of transplanted cells

• Mechanism of action??

• Not tested in appropriate animal model

A path to the clinic: preclinical studies

Obtain proof-of-principle data in an animal model of epilepsy

• Generation of inhibitory interneurons

• Functional integration following transplantation

• Disease-modifying activity (DMA) against spontaneous seizures and related co-morbidities

American Epilepsy Society | 2013 Annual Meeting

How to make an interneuron for cell therapy

Source: embryonic medial ganglionic eminence (MGE) • neuron expressing GABA • migration in host brain • mature firing properties • integration in host circuitry

E13.5 mouse

MGE cells migrate widely in host brain

• P2 transplant (up to 5 mm)

• P60 transplant (up to 2 mm)

• cortex, hippocampus, amygdala

MGE cells enhance inhibition in host brain

• fetal MGE progenitor cells

• IPSC onto host pyramidal

• cortex and hippocampus

Optogenetic manipulation of MGE-derived interneurons following transplantation

Robert F. Hunt III

MGE cells in a genetic form of epilepsy

• Kv1.1 null mice

• P2 MGE transplant

• 96% seizure reduction

Baraban SC, Southwell DG, Estrada RC, Jones DL, Sebe JY, Alfaro-Cervello C, Garcia-Verdugo JM, Rubenstein JL, Alvarez-Buylla A. Proc. Natl. Acad. Sci. USA 2009

MGE cells in an acquired form of epilepsy

• pilocarpine model of TLE

• adult MGE transplant

• 92% seizure reduction

Hunt RF, Girskis KM, Rubenstein JL, Alvarez-Buylla A, Baraban SC. Nat. Neuroscience 2013

How to make a neuron: induced neural cells

Yang N, Ng YH, Pang ZP, Sudhof TC, Wernig M. Cell Stem Cell 2011

• defined transcription factors direct cell fate

Features of an ideal human MGE cell line

• GABA interneuron specificity – PV, SOM, NPY

• Robust migration

• Functional integration

• Safety

• Efficacy

American Epilepsy Society | 2013 Annual Meeting

Some currently available human stem cell lines

1. Sox1-expressing embryonic stem cells (ESC)

2. Nkx2.1-expressing embryonic stem cells (ESC)

3. induced pluripotent stem cells (iPSC) from Dravet patients

Maisano et al. J Neurosci 2012; Liu et al. Nat Biotechnol 2013; Maroof et al. Cell Stem Cell 2013; Nicholas et al. 2013; Liu et al. Ann Neurol 2013; Jiao et al. Hum Mol Genet 2013; Shirasaka et al. Mol Brain 2013

Human SC-derived interneurons in vitro

Kevin Ess

Stewart Anderson

Lorenz Studer

Arturo Alvarez-Buylla, John Rubenstein

Arnold Kriegstein

Lori Isom

Jack Parent

GABA / b-tubulin III

Su-Chun Zhang

Potential problems: Slow differentiation

• NKX2.1::GFP human ES cells

• FACS sorted and transplanted into neonatal cortex

• 6 wk - 3 mo. post-transplantation: “undifferentiated appearance . . . often tipped by growth cones”

Maroof AM, Keros S, Tyson JA, Ying SW, Ganat YM, Merkle FT, Liu B, Goulburn A, Stanley EG, Elefanty AG, Widmer HR, Eggan K, Goldstein PA, Anderson SA, Studer L. Cell Stem Cell 2013

Potential problems: Slow functional maturation

• NKX2.1::GFP human ES cells

• FACS sorted/neonatal cortex

• 7 months post-transplant

Nicholas CR, Chen J, Tang Y, Southwell DG, Chalmers N, Vogt D, Arnold CM, Chen YJ, Stanley EG, Elefanty AG, Sasai Y, Alvarez-Buylla A, Rubenstein JL, Kriegstein AR. Cell Stem Cell 2013

Potential problems: Teratocarcinoma

• undifferentiated hES cells

• hippocampus transplant

• Sox1:GFP::Ubi:RFP cells

Germain ND, Hartman NW, Cai C, Becker S, Naegele J, Grabel LB. Cell Transplant 2012

How do we address these problems?

• Learn more about the properties of fetal human MGE cells

• Improve methods or techniques for generating hMGE cells

• Develop and characterize more appropriate animal models for evaluation of human MGE cells

Properties of human MGE cells

• Subcortical origins of human and monkey neocortical interneurons. Ma et al. Nat Neuroscience 2013

• Non-epithelial stem cells and cortical interneuron production in the human ganglionic eminences. Hansen et al. Nat Neuroscience 2013

“Cerebral organoids” as MGE donors?

• Cerebral organoids model human brain development and microcephaly. Lancaster et al. Nature 2013

• Self-organization of axial polarity, inside-out layer pattern, and species progenitor dynamics in human ES cell-derived neocortex. Kadoshima et al. Proc Natl Acad Sci USA 2013

Human stem cell therapies that are showing some promise (but not epilepsy, yet)

• Neural stem cell engraftment and myelination in the human brain. [leukodystrophy] Gupta et al. Sci Transl Med 2012 • Central nervous system stem cell transplantation for children with neuronal ceroid lipofuscinosis. J Neursurg Pediatr. 2013

HuCNS-SC oligodendrocytes, neurons and astrocytes

A path to the epilepsy clinic

Obtain IND enabling data with a human MGE cell line

• Disease Modifying Activity (DMA) in at least one animal model of epilepsy

• Safety and “dosing” information

• Cryopreservation

• Device for delivery of human MGE cells

American Epilepsy Society | 2013 Annual Meeting

(Future) Impact on Clinical Care and Practice

1987-1990

Tissue grafts transiently suppress evoked seizures

(Future) Impact on Clinical Care and Practice

2013 1987-1990

Tissue grafts transiently suppress evoked seizures

Mouse MGE cells suppress spontaneous seizures & reverse comorbidities

(Future) Impact on Clinical Care and Practice

2013 1987-1990 2025

Tissue grafts transiently suppress evoked seizures

Mouse MGE cells suppress spontaneous seizures & reverse comorbidities

Human MGE cells are safe and suppress seizures in patients