NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in genes that encode receptor subunits are associated with several intractable neurodevelopmental disorders, including developmental and epileptic encephalopathy (DEE). To extend understanding of these intractable childhood diseases, we have begun to model these variants in laboratory mice. Models studied so far are a constitutive knockin for GRIN2A variant p.Ser644Gly (S644G) based on one case, and a GRIN2D variant, p.Val667Ile (V667I), noted in at least three unrelated cases. Homozygous and heterozygous Grin2a S644G mice exhibit altered hippocampal morphology at 2 weeks, and homozygotes exhibit lethal tonic-clonic seizures in week 3. Heterozygotes have a normal lifespan without spontaneous seizures, but display a variety of distinct features including resistance to electrically induced limbic seizures, as well as hyperactivity and repetitive and reduced anxiety behaviors. Multielectrode recordings of mutant neuronal networks reveal hyperexcitability and altered bursting and synchronicity of both mutant genotypes. When expressed in heterologous cells, mutant receptors exhibit enhanced NMDAR agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. Consistent with this, NMDAR-mediated synaptic currents in hippocampal slices from mutant mice show a prolonged deactivation time course. Standard antiepileptic drug monotherapy was ineffective in the patient, but combined treatment of NMDAR antagonists with antiepileptic drugs substantially reduced the seizure burden albeit without appreciable developmental improvement. Chronic treatment of homozygous mutant mouse pups with NMDAR antagonists delayed the onset of lethal seizures but did not prevent them. Grin2d V667I knockin mice are at an earlier stage of study. In contrast to Grin2a S644G, V667I heterozygous mice are severely impaired and suffer from lethal tonic-clonic seizures with an onset from about 18 days to 3 months. The impairment precludes natural mating such that the line needs to be propagated by ovary transplantation. Young heterozygotes adults in video-EEG also exhibit very frequent interictal epileptiform spiking and spike-wave discharge activity, resembling absence seizures. Preliminary histology shows significant pyknotic nuclei appearing to evidence cell death in the cerebral cortex. Pup developmental milestones, while not evidencing significant developmental delay, show unusual features including excessive maternal separation induced pup vocalization. Further studies are ongoing. Together these efforts illustrate the power of modelling severe neurodevelopmental seizure disorders using multiple experimental modalities and suggest their utility in identifying and evaluating new therapies.

1. Hammer Bldg IGM
4-5-6 floors
Mouse Models of GRIN Gain-Of-Function Genetic Variants
Wayne N. Frankel, Ph.D.
Institute of Genomic Medicine (IGM)
Department of Genetics & Development
Columbia University Irving Medical Center, NY, NY
© Wayne N. Frankel, Ph.D.

2. Columbia/IGM
Frankel lab
David Goldstein
Michael Boland
Jennifer Gelinas
Yueqing Peng
Emory University
Stephen Traynelis & colleagues
University of Vermont
Matthew Weston
The Jackson Laboratory
Cat Lutz
Boston Children’s/Harvard
Anapurna Poduri & colleagues
Nationwide Children’s/Ohio St.
Scott Harper
• Explore mouse models of severe childhood epileptic encephalopathy
• Can we obtain relevant in vivo phenotypes when modeling the mouse version of disease?
• Including and beyond seizures
• Establish studies across several experimental platforms
• in vitro, in vivo, ex vivo
• Phenotypes & mechanisms
• Intervention feasibility
• Test predictable or invent new interventions
• Evaluate in ≥1 platform
• Test efficacy vs. ‘standard of care.’
Objectives of our research
© Wayne N. Frankel, Ph.D.

3. Human genetics,
genomics
David Goldstein
Erin Heinzen
Mouse models:
seizures, sleep
Wayne Frankel
Jennifer Gelinas
Yueqing Peng
Mouse models:
neurobehavior
Mu Yang, MNBC
Fibroblasts hiPSCs
O S
K M
Neural Networks
Integration-free
reprogramming
Neuronal
Differentiation
Electrophysiology
Multielectrode Array
Dermal biopsy
Genetic mouse models
Small Molecule
Screening
Genome Editing
(CRISPR/Cas9)
Gene Expression
Analyses
Patient-specific
mutations
Seizure monitoring
Seizure threshold testing
Comprehensive behavior
monitoring
Drug Pharmacology
Genetic
variant
detection
Patients
Clinical partners
Neurogenetics research in the IGM - a precision medicine ‘ecosystem’
Cellular
models
Michael Boland
© Wayne N. Frankel, Ph.D.

4. But please keep in mind:
• Mice are mice, and people are people
• Clinical features not expected to be identical (and they are not!)
• Beyond speech deficits
• Variation in type and prominence of clinical features and gene dosage impact
• However, most genes and many basic neurological functions are very highly conserved
• Same gene -> neuron function -> brain-wide function -> mouse version of disease
= Best possible animal model of genetic disease, including neurological
(despite clear differences between species, and the fact that we are only just
beginning to get good at assessing phenotypes during “mouse childhood”)
© Wayne N. Frankel, Ph.D.

5. A2M BHLHE22 CHD4 DIP2C FETUB HDAC4 LANCL2 MYO5A PACS2 PTPRO RXFP1 STK36 TTN
AAK1 BMP2 CHIA DISP1 FLG HECW2 LCE1A MYO7B PAK6 PTPRT RYR2 STX1B TTYH1
ABCA2 BMS1 CLDN19 DNAH7 FLNA HFE LDLRAD1 MYOM3 PALLD PURA RYR3 STXBP1 TUBB2A
ABCB9 C16orf62 CLIC5 DNAH9 FLNC HIPK3 LEKR1 N6AMT1 PAQR8 PWWP2A SAFB2 SVOPL UBQLN4
ACOT4 C17orf53 CNTN5 DNAJC6 FLRT1 HIST1H2BD LEMD2 NBAS PASK QRSL1 SCAF4 SYNE2 UHRF1BP1L
ADAM21 C18orf25 COL4A4 DNM1 FOCAD HIST2H2BE LETM1 NBEA PCDHB13 RAB5C SCN1A SYTL5 UNC5CL
ADAMTSL4 C1orf123 COL7A1 DSG2 FRAT2 HLTF LIN7A NCBP1 PDCL2 RAD54L2 SCN2A TAAR2 USP7
AGPAT3 C1orf56 COQ3 DTYMK FRMD4A HNRNPH1 LRP1 NCOR2 PDIK1L RAET1L SCN8A TAF1 UTRN
AHCY C1QTNF6 CPAMD8 EDEM1 G3BP1 HNRNPU LRP4 NEDD4L PHF21A RALGAPB SCYL1 TAS2R4 VPS37A
AKAP6 C3orf22 CR2 EMILIN3 GABBR2 HRG LUC7L3 NEDD9 PHIP RALGPS1 SDCBP2 TCF4 WDFY2
AKR1C4 C4orf37 CREBBP EPHB1 GABRA1 HSF2 MAML3 NETO2 PIGS RANBP17 SELRC1 TCTE3 WDR1
ALG13 C5orf22 CRTAC1 ERG GABRB1 HSPG2 MAN1A2 NFASC PIK3AP1 RANGAP1 SERPINC1 TEP1 WDR19
ALMS1 C6orf222 CSMD2 ETNK2 GABRB3 IFT172 MAP3K8 NFE2L1 PIKFYVE RARS SETX TET3 WDR45
ALS2CL CACNA1A CSNK1E ETS1 GAS2 IQSEC2 MAPK8IP1 NFRKB PITX1 RASIP1 SGK223 TEX15 WDR82
ANK3 CACNA1E CTTNBP2NL EXOSC2 GCM2 ITGAM MAST1 NIPA1 PLA1A RBM12 SKA3 THAP4 WHSC1L1
ANKRD12 CAMK4 CUBN EXPH5 GFM2 ITGB4 MCM3 NLGN2 PLCG2 RBM45 SLAMF1 THOC2 WRN
ANKRD24 CANT1 CUL2 FAM102A GLB1L3 ITPR1 MCM7 NLRP11 PLXNA1 RCL1 SLC16A3 TIFA XPO1
ANKRD50 CASP14 CUX2 FAM116B GLIS3 KCNB1 MEOX2 NLRP5 PLXNB1 RD3 SLC1A2 TMPRSS5 YPEL4
AP3S2 CASP9 CXXC11 FAM133B GLUL (KCNQ2) MIOX NLRP8 PNMAL1 RET SLC25A13 TNKS2 YWHAG
ARFGEF1 CASQ1 CYP2U1 FAM134A
GNAO1
GNB1 (KCNQ3) MKLN1 NOLC1 PPP1R3B RFX3 SLC26A11 TNNI3K ZBTB40
ARRDC1 CCDC125 DAO FAM21C GPR108 KCNT1 MLL NOTUM PPP3CA RGS14 SLC26A8 TPTE2 ZC3H3
ARHGEF9
ASH1L CDC25B DBP FAM50A
GPR128
GPR98 KDR MLL2 NPAT PPP6R2 RHOG SLC35A2 TRIM29 ZFHX3
ASXL1 CDHR2 DCX FAM63B GRAMD2 KIAA0913 MMP27 NR1H2 PRDM12 RIOK3 SLC5A10 TRIM32 ZNF248
ATAD2B CDKL5 DDX50 FAM86C1 GRIN1 KIAA1324L MRS2 NTSR2 PRDM4 RNF186 SLCO1B7 TRIM8 ZNF282
ATIC CDS2 DDX58 FARSA GRIN2A KIAA2018 MSANTD1 OR10S1 PRG3 RP1L1 SMG9 TRIO ZNF354C
ATP2B4 CELA3B DECR2 FASN
GRIN2B
GRIN2D KLHL11 MTOR OR2F2 PRKX RRP1B
SZT2
SMURF1 TRRAP ZNF572
B3GNT4 CELSR1 DHDDS FBXL4 GTF2B KMT2B MTRF1 OR52E8 PRR19 RTKN2 SNX30 TSNAXIP1 ZNF839
BCL2L13 CEP55 DHTKD1 FBXO41 HBS1L KNDC1 MVK OSBPL5 PSD3 RTN1 SORBS3 TSPYL1 ZNFX1
BCLAF1 CHD2 DIAPH3 FCGR2B HCK KRT34 MYH6 OSBPL7 PTEN RTP1 SP TTC16 ZSCAN2
BEST2 DIP2B FERMT3 HCN4 KRTAP1-3 MYO3A OXA1L PTK2B RUVBL2 SPG7 TTF1 ZSCAN21
Origins of our research – breakthroughs in identifying putative genetic variants for epilepsy
Epi4k
EPGP
EpiGen
& other
Genome
sequencing
groups
& consortia
© Wayne N. Frankel, Ph.D.

6. Gene and variant Whole animal phenotypes? Histological phenotypes? Neuron culture phenotypes? Therapy efficacy studies begun?
Grin2aS644G
NMDA receptor/ion channel
Seizure & behavior Hippocampal atrophy Yes Yes (NMDAR antagonists)
Grin2dV664I
NMDA receptor/ion channel
Seizure (behavior NDY) Cell death NDY Yes (NMDAR antagonists)
Gnb1K78R
G-protein b1 subunit
Seizure & behavior
No
Yes
Yes (antiepileptics)
Kcnt1Y796H
“Slack” K+ ion channel, mild
Seizure & behavior No Yes Excluded proposed therapy
Kcnt1condR428Q
“Slack” K+
ion channel, severe
Seizure (behavior NDY) NDY NDY NDY
Arhgef9G55A
GEF & scaffold for inhibitory synapses
Seizures NDY; startle
Protein aggregates, selective
neuronal death
NDY NDY
Arfgef1fs
GEF and trans-Golgi protein
Seizure (↓ threshold only)
and pup milestones
Hippocampal atrophy No NDY
Iqsec2fs
GEF & scaffold for excitatory synapses
Seizure & behavior Hippocampal ‘swelling’ NDY NDY
Ppp3cafs*
Calcineurin: Ser/Thr prot. phosphatase
Seizure (behavior NDY) NDY NDY NDY
Stxbp1-/+
MUNC-18: synaptic vesicle exocytosis
Seizure & behavior (no) Yes NDY
Dnm1Ftfl/Ftfl
Dynamin 1: synaptic vesicle
endocytosis
Seizure & behavior
Cell death, dendritic
morphology
Yes Yes (mRNA silencing)
Genetically diverse (not just ion channels) mouse models under study in our group

7. 4 mos 9 mos
Various Various
(incl. atyp.
absence)
No No
NMDAR antagonists
lower sz. freq.
Profound
Hypotonia Spastic
Ocularmotor apraxia
Non-verb.
Non ambul.
LBD
M1
M3
ABD
ATD
M4
M2
*
d GluN1/GluN2A Tetramer
S644
Transmembrane
Domains
Agonist
Binding
Domain
Amino
Terminal
Domain
COOH
GluN2A
Out
In
Agonist
*
Human GluN2A (620) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEFVD (660)
Human GluN2B (621) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEYVD (661)
Human GluN2C (618) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEQYID (658)
Human GluN2D (648) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEEYVD (688)
Human GluN1 (622) GAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEE (662)
Val667Ile
(GluN2D)
Ser644G
(GluN2A)
*
Two NMDA receptor subunit variants causing early onset epileptic encephalopathy
S644G V667I
Onset:
Seizures:
AED ther. resp.:
Other drugs:
Devel delay:
Movement:
Other:
GRIN2A
(GluN2A)
GRIN2D
(GluN2D) NMDAR = Ligand-gated (glycine, glutamate) selective cation channel (Na+, Ca2+)
Anapurna Poduri et al., Boston Children’s Hospital Stephen Traynelis et al., Emory University

8. a%ent’s and muta%on’s informa%on. (a) (b) pa%ent’s informa%on. (c) Schema%c topology
ents a GluN2A subunit (asterisk notes the posi%on of the S644G muta%on). (d) A model of
A subunit shown as space fill built from the GluN2B crystallographic data. The red asterisk
artoon indica%ng the domain arrangement of a GluN2A subunit shows the posi%on of
the transmembrane domain M3 (TM3), a cri%cal domain that may influence the channel
LBD
M1
M3
ABD
ATD
M4
M2
*
d GluN1/GluN2A Tetramer
S644
Transmembrane
Domains
Agonist
Binding
Domain
Amino
Terminal
Domain
COOH
GluN2A
Out
In
Agonist
c GRIN2A
ATD S1 S2 CTD M1
M2
M3 M4
*
*
Human GluN2A (620) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEFVD (660)
Human GluN2B (621) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEYVD (661)
Human GluN2C (618) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEQYID (658)
Human GluN2D (648) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEEYVD (688)
Human GluN1 (622) GAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEE (662)
MGI 958 Grin2a
Exon 10
WT
S644G
429kb
Chr. 16
GCA AGT TAC
GCA GGT TAC
A S Y
A G Y
CRISPR/Cas9 JAX GET oligo-directed HDR
221 1 1 2
221 1 1 2
221 1 2
221 1 2
1 1 1 2
221 1 1 2
221 1 1 2
221 1 2
221 1 2
1 1 1 2
1. All homozygotes have lethal seizures by PND17
Days postnatal
0 10 20 30
0
50
100
Age postnatal (days)
%Survival
+/+
S644G/+
S644G/S644G
%Survival
Grin2aS644G knockin mouse model
Cat Lutz, Aamir Zuberi, JAX Center for Precision Genetics Frankel lab, Columbia
Most overt features
2. Heterozygous mothers inattentive to pups
3. Heterozygous mice are hyperactive

9. HIP
COR
HIPHIP
CA1 CA2
DG
UL
CA1
DL
L1
VZ
+/+ +/S644G S644G/S644Ga
HIP CA2
DG
CA1 HIP CA2
DG
CA1
Grin2aS644G heterozygous and homozygous mice have hippocampal atrophy at
postnatal day 14 (2-3 days before lethal seizures)
JJ Teoh, Frankel lab, Columbia
Heterozygous adults resistant to electrically induced limbic seizure
+/+ S644G/+
0
5
10
15
20
iRMSCurrent(mA)
****
iRMScurrent(±1SD)
© Wayne N. Frankel, Ph.D.

10. +/
+
Baseline 80 90 100 110 120
0
100
200
300
400
Startleresponse(Vmax)
+/+
S644G/+
Stimulus (dB)
*
***
***
***N=25
N=22
*
circling repetititve exploring resting
0
20
40
60
80
100
Percent(%)
+/+
S644G/+
N=12
N=11
***
**
Stimulus (dB)
WT Mutant
0
10
20
30
40
50
60
70
80
90
Threshold
0
20
40
80
60
ABRthreshold(dB) p<0.05
Acoustic startle Repetitive behaviors Self-grooming
p<0.1
p<0.05
p<0.001
10 20 30 40 50 60
500
1000
1500
2000
Recording time (min)
Distancetraveled(cm)
+/+ female (N=9) S644G/+ female (N=13)
+/+ male (N=12) S644G/+ male (N=12)
10 20 30 40 50 60
0
100
200
300
Recording time (min)
Timespentincenter(s)
+/+ female S644G/+ female
+/+ male S644G/+ male
Open field (total ambulation) Open field (center time)
females males both
0
20
40
60
80
Time(s/10mins)
+/+
S644G/+
*
4 4 7 9 11 13
Grin2aS644G heterozygous adults have a variety of behavioral phenotypes
Ayla Kanber, Ariadna Amador, Frankel lab, Columbia

11. 11
1E-8 1E-7 1E-6
0
20
40
60
80
100
0.01 0.1 1 10 100
0
20
40
60
80
100
1E-3 0.01 0.1 1 10
0
20
40
60
80
100
S644G
WT 2A
Glutamate, µM
MaximalResponse,%
Glycine, µM
S644G
WT 2A
MaximalResponse,%
H+, M
MaximalResponse,%
500 ms
0.1 normalized
S644G
WT 2A
▼
glutamate
GluN1/GluN2A-S644G Heterologous Expression System
S644G
WT 2A
Functional properties of receptor-channel:
GRIN2A (GluN2A) -S644G enhances virtually all aspects of receptor function
Yuan, Traynelis labs, Emory© Wayne N. Frankel, Ph.D.

12. 12
+ / + S 6 4 4 G / +
0
6 0
1 2 0
1 8 0
tweighted(ms)
N M D A R S y n a p t i c D e c a y
N = 1 5 N = 9
* * *
Evoked CA1 EPSC from S644G
200 ms
50 pA
WT
S644G +/-
0.1
0.2
100 ms
S644G mutation prolongs NMDAR synaptic time course (more excitable)
200 ms
50 pA
Bhattacharya, Camp - Yuan/Traynelis labs, Emory
10
100
1000
GluN2AC1/GluN2AC2
GluN2AC1-S644G/GluN2AC2
GluN2AC1-S644G/GluN2AC2-S644G
glutamate
300 ms
tweighted(ms)
GluN2AC1/
GluN2AC2
GluN2AC1-S644G/
GluN2AC2-S644G
GluN2AC1-S644G/
GluN2AC2
(14) (8) (9)
GluN1/GluN2A-S644G HEK cells
© Wayne N. Frankel, Ph.D.

13. Electrodes embedded in cell culture plate (48 well – 16 electrodes per well)
Advantages (vs. single cell patch-clamp recordings):
• Observe populations of neurons rather than single cells
• Non-invasive method to record neuronal network activity
• Monitor network development/establishment
• Test several experimental conditions in parallel
• Medium-throughput allows for small-scale compound screening/testing
Neurons on MEA
Multiwell MEA
Network firing properties 1˚ neuron culture in Multielectrode Array (MEA)
© Wayne N. Frankel, Ph.D.

14. Grin2a-S644G enhances spike and network burst firing in 1˚ cortical neuron culture – MEA
Christopher Bostick, Daniel Krizay, Goldstein/Boland labs, Columbia
+/+ S644G/S644G
0 20 40 60
1
16
S644G/+
0 20 40 60
1
16
Electrodes
0 20 40 60
Time (s)
1
16
p < 0.0001 each DIV
5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9
0
1 0
2 0
3 0
D a y s In V itro (D IV )
Burst/min
+ /+
S 6 4 4 G /S 6 4 4 G
S 6 4 4 G /+
P e rm p -v a lu e : < 0 .0 0 1 + /+ v s S 6 4 4 G /+ a n d S 6 4 4 G /S 6 4 4 G ,
0 .2 7 9 S 6 4 4 G /+ v s S 6 4 4 G /S 6 4 4 G
5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9
0 .0
0 .1
0 .2
0 .3
D a y s In V itro (D IV )
MeanFrequencyinBurst
+ /+
S 6 4 4 G /S 6 4 4 G
S 6 4 4 G /+
P e rm p -v a lu e : < 0 .0 0 1 + /+ v s S 6 4 4 G /+ a n d S 6 4 4 G /S 6 4 4 G ,
0 .6 4 S 6 4 4 G /+ v s S 6 4 4 G /S 6 4 4 G
p < 0.0001 each DIV
5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9
0
5
1 0
1 5
D a y s In V itro (D IV )
MFR(Hz/AE)
+ /+
S 6 4 4 G /S 6 4 4 G
S 6 4 4 G /+
P e rm p -va lu e : < 0 .0 0 1 + /+ vs S 6 4 4 G /+ a n d
S 6 4 4 G /S 6 4 4 G , 0 .6 4 S 6 4 4 G /+ v s S 6 4 4 G /S 6 4 4 G ,
p < 0.0001 each DIV
1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9
D a y s In V itro (D IV )
+ /+
S 6 4 4 G /S 6 4 4 G
S 6 4 4 G /+
: < 0 .0 0 1 + /+ vs S 6 4 4 G /+ a n d
.6 4 S 6 4 4 G /+ v s S 6 4 4 G /S 6 4 4 G ,
Mean firing rate # Burst per minute Mutual information
Mutual Information
© Wayne N. Frankel, Ph.D.

15. Memantine
Dextromethorphan
Topiramate
Levetiracetam
Valproic acid
Zonisamide
Max dose
No.seizurespermonth
0
50
100
150
200
250
Mar-15
May-15
Jul-15
Sep-15
Nov-15
Jan-16
Mar-16
May-16
Jul-16
Sep-16
Nov-16
Jan-17
Mar-17
May-17
Jul-17
Sep-17
Monthly Seizure Count
GRIN2A S644G patient seizure freq. lowered by NMDAR antagonist & AED combined therapy
(“comorbid” behaviors unimproved)
Anapurna Poduri, Jurrian
Peters, Heather Olson,
Harvard Univ/Boston
Children’s Hospital
© Wayne N. Frankel, Ph.D.

16. MFR(Hz)%Baseline
-7 -6 -5 -4 -3
0
2 0
4 0
6 0
8 0
1 0 0
G lu N 2 A C1 /G lu N 2 A C2
G lu N 2 A C1 -S 6 4 4 G /G lu N 2 A C2
G lu N 2 A C1 -S 6 4 4 G /G lu N 2 A C2 -S 6 4 4 G
L o g [M e m a n tin e ]
Response(%Control)
-7 -6 -5 -4 -3
0
2 0
4 0
6 0
8 0
1 0 0
G lu N 2 A C1 /G lu N 2 A C2
G lu N 2 A C1 -S 6 4 4 G /G lu N 2 A C2 -W T
G lu N 2 A C1 -S 6 4 4 G /G lu N 2 A C2 -S 6 4 4 G
L o g [D e x tro m e th o rp h a n ]
Response(%Control)
MFR(Hz)%Baseline
Memantine (µM) Dextromethorphan (µM)
%survival
Log [Memantine] (µM) Log [Dextromethorphan] (µM)
Response(%ofcontrol)
Response(%ofcontrol)
Age (days postnatal)
0 10 20 30
0
50
100
Age (days postnatal)
Percentsurvival
Vehicle (N=24)
Radiprodil (N=15)
Nuedexta (N=17)
Dextromethorphan (N=16)
Quinidine (N=11)
0.01 0.1 1 10 100
0
50
100
150
Memantine (µM)
MFR(Hz)%Untreated
+/+
S644G/+
Non-treated
S644G/S644G
0.1 1 10
0
50
100
150
Dextromethorphan (µM)
MFR(Hz)%Untreated
+/+
S644G/+
S644G/S644G
Non-treated
0.01 0.1 1 10 100
0
50
100
150
Memantine (µM)
MFR(Hz)%Untreated
+/+
S644G/+
Non-treated
S644G/S644G
Response to FDA-approved NMDAR antagonists in vitro, ex vivo and in vivo
Weiting Tang, Traynelis/Myers/Yuan labs, Emory
Christopher Bostick, Goldstein/Boland labs, Columbia
Ariadna Amador, Ayla Kanber, Frankel lab, Columbia
Nuedexta prolongs survival by 30%
© Wayne N. Frankel, Ph.D.

17. Summary: Grin2a S644G model
Strong in vitro, in vivo and ex vivo phenotypes
Seizures
Hyperactive, hypoanxious, repetitive behaviors, poor maternal care
Mostly consistent with each other
Some unusual (e.g. resistance to limbic seizures)
NMDAR targeted drug studies mimic partial rescue seen in human
Does not mitigate developmental delay (treated mice still very small)
© Wayne N. Frankel, Ph.D.

18. 4 mos 9 mos
Various Various
(incl. atyp.
absence)
No No
NMDAR antagonists
lower sz. freq.
Profound
Hypotonia Spastic
Ocularmotor apraxia
Non-verb.
Non ambul.
LBD
M1
M3
ABD
ATD
M4
M2
*
d GluN1/GluN2A Tetramer
S644
Transmembrane
Domains
Agonist
Binding
Domain
Amino
Terminal
Domain
COOH
GluN2A
Out
In
Agonist
*
Human GluN2A (620) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEFVD (660)
Human GluN2B (621) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEYVD (661)
Human GluN2C (618) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEQYID (658)
Human GluN2D (648) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEEYVD (688)
Human GluN1 (622) GAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEE (662)
Val667Ile
(GluN2D)
Ser644G
(GluN2A)
*
Two NMDA receptor subunit variants causing early onset epileptic encephalopathy
S644G V667I
Onset:
Seizures:
AED ther. resp.:
Other drugs:
Devel delay:
Movement:
Other:
GRIN2A
(GluN2A)
GRIN2D
(GluN2D) NMDAR = Ligand-gated (glycine, glutamate) selective cation channel (Na+, Ca2+)
Anapurna Poduri et al., Boston Children’s Hospital Stephen Traynelis et al., Emory University
3 4 children
have the
same variant

19. Grin2dV667I/+ heterozygotes experience lethal seizures b/w PND22-50
JAX: Heroic approach to line maintenance: ovary transplantation
Sabrina Petri, Frankel lab, Columbia
Cat Lutz lab, JAX
In our hands, memantine ad lib
in the drinking water does not
significantly mitigate lethal seizures – but its
difficult to control dosage or confirm degree of
brain exposure.
© Wayne N. Frankel, Ph.D.

20. WT32
WT33
WT34
Het35
Het36
Het40
Het39
F
F
F
F
M
F
M
Seizure phenotypes of Grin2dV667I/+ mice seen as early as 17 days of age
1. Continual spike-wave discharge-like seizure activity in Grin2d V667I mice
2. Single, lethal tonic-clonic seizure
Sabrina Petri, Frankel lab, Columbia
© Wayne N. Frankel, Ph.D.

21. Grin2D V667I – abnormal motor/coordination
Wildtype
hindlimbs
splayed
Heterozygote
hindlimbs
clasped
© Wayne N. Frankel, Ph.D.

22. Het/Hom weigh less than
wt as they grow
No difference Different at P7
No difference No difference Different at P6
Grin2d V667I abnormal pup behaviors at a very early age postnatal
JJ Teoh, Frankel lab, Columbia
Postnatal day 3!
© Wayne N. Frankel, Ph.D.

23. Summary and plans: Grin2aV667I model
Early days in our studies
Challenge to overcome breeding obstacle (due to lethal seizures of otherwise fertile adults)
Continue with ovarian transplantation, but conditional model (“floxxed”) – underway at JAX
Seizure phenotypes – striking
Determine age-of-onset and progression of continual spike-wave seizures
Histopathology
Determine age-of-onset of cell death, cell types
Cellular physiology
Whole-cell recordings in neuron culture (Columbia) & acute slices (Emory)
Multielectrode array mass culture recordings
Cellular etiology (is defect in interneurons rate-limiting for disease?)
Ideally want conditional mutation for this
Treatment
Continue to explore memantine + (chronic treatment challenges in mice)
Genetic therapy (e.g. AAV9-mediated mutation-specific RNA elimination?)
© Wayne N. Frankel, Ph.D.

24. Novel gene therapy approach: Dnm1Fitful
mouse model for Dnm1 epileptic encephalopathy
GTPase Middle PHD GED PRD
1 314 499 631 746 864
G43S
S45N T65N
G139V
Q148R
A177P
K206N
K206G R237W
S238I
I289F G346V
G359A
G359R
G373K
H396D
G397D
N363_R364insLP K535E
A408T Ftfl
Mutant mouse discovered
spontaneously at Jackson Lab ca. 2008
Virginia Aimiuwu,
Ph.D. candidate• Dnm1Ftfl/Ftfl homozygotes have severe seizures, usually lethal by 3rd week
• Modeling EE
• Various neurodevelopmental and behavioral deficits
We previously showed seizures due to inhibitory neuron mutant expression,
but neurobehavioral comorbidities due to excitatory neuron mutant expression
© Wayne N. Frankel, Ph.D.

25. DNM1 codes for a protein involved in endocytosis and synaptic vesicle recycling
Schmid and Frolov 2011
• Dnm1Ftfl molecules interfere with DNM1 multimolecular assembly
© Wayne N. Frankel, Ph.D.

26. Dnm1a specific miRNA construct
Scott Harper, Nationwide Children’s Hospital Center for Gene Therapy
Novel gene therapy approach: inactivate (degrade) mutant mRNA
© Wayne N. Frankel, Ph.D.

27. miDnm1A-4 shows the highest efficacy of Dnm1a knockdown in vitro
• miDnm1A-1: 84%
• miDnm1A-2: 64%
• miDnm1A-3: 80%
• miDnm1A-4: 95%
Scott Harper, Nationwide Children’s Hospital Center for Gene Therapy
© Wayne N. Frankel, Ph.D.

28. Package and deliver miRNA construct in
adeno-associated virus, AAV9 (neurotropic subtype)
Schultz and Chamberlain., 2009
© Wayne N. Frankel, Ph.D.

29. Experimental design
scAAV9-miDnm1a
icv injection
E10 E15 E20 P2 P4 P6 P8 P10 P12 P14 P16 P18 P20P0
Neurogenesis
Synaptogenesis
growth delay
Ataxia
Wobbly gait
Seizures
Hypotonia Lethal seizures
D
nm
1a
nm
1b
D
nm
1
0
2
4
6
ΔCTofDnm1relativemRNA
+/+ eGFP
+/+ miDnm1a
Virginia Aimiuwu, Frankel lab, Columbia
© Wayne N. Frankel, Ph.D.

30. 0 10 20 30
0
50
100
PND
Percentsurvival
Survival
Ftfl/Ftfl
Ftfl/Ftfl: 1x1010 vg miDnm1a
Ftfl/Ftfl: 1.85x1011 vg miDnm1a
Ftfl/Ftfl: 3.25x1011 vg miDnm1a
+/Ftfl untreated
+/+
0 10 20 30
0
5
10
15
20
25
PND
Weight(g)
Growth
Ftfl/Ftfl
Ftfl/Ftfl: 1.85x1011 vg miDnm1a
Ftfl/Ftfl: 3.25x1011 vg miDnm1a
+/Ftfl untreated
+/+
0 10 20 30
0
PND
Ftfl/Ftfl
Ftfl/Ftfl: 1x1010 vg miDnm1a
Ftfl/Ftfl: 1.85x1011 vg miDnm1a
Ftfl/Ftfl: 3.25x1011 vg miDnm1a
+/Ftfl untreated
+/+
0 10 20 30
0
50
100
PND
Percentsurvival
F2 Hybrid Survival
+/+
+/+: 5.2x1011 vg
miDnm1a
Ftfl/Ftfl: 5.2x1011 vg
eGFP
Ftfl/Ftfl: 5.20x1011 vg
miDnm1a
0 10 20 30
0
5
10
15
20
25
PND
Weight(g)
F2 Hybrid Growth
+/+
+/+: 5.2x1011 vg miDnm1a
Ftfl/Ftfl: 5.2x1011 vg eGFP
mRNA silencing therapy in Dnm1Ftfl/Ftfl neonates significantly mitigates lethal seizures AND
developmental delay (and on two different strain backgrounds)(B6JxFVB)F2
hybrid
B6Jinbred
Growth Survival
Virginia Aimiuwu, Frankel lab, Columbia
Rescues features previously shown to
depend on gene defect in excitatory neurons
……inhibitory neurons
© Wayne N. Frankel, Ph.D.

31. 2 4 6 8 10 12
0
2
4
6
8
PND
weight(g)
Growth
Ftfl/Ftfl Treated: 3.25x1011 vg
Ftfl/Ftfl
+/+
3 5 7 9 11 13
0
10
20
30
40
PND
Time(S)
Negative geotaxis 90o
Ftfl/Ftfl: 3.25x1011 vg miDnm1a
Ftfl/Ftfl
+/+
3 5 7 9 11 13
0
10
20
30
40
PND
Time(S)
Negative geotaxis 180o
Ftfl/Ftfl: 3.25x1011 vg miDnm1a
Ftffl/Ftfl
+/+
It also rescues deficiency in some developmental performance tasks
Virginia Aimiuwu, Frankel lab, Columbia
© Wayne N. Frankel, Ph.D.

32. Promising, but why isn’t rescue complete?
treatment
E10 E15 E20 P2 P4 P6 P8 P10 P12 P14 P16 P18 P20P0
Neurogenesis
Synaptogenesis
growth delay
Ataxia
Wobbly gait
Seizures
Hypotonia Lethal seizures
Human window of opportunity should be longer
Quality of life improvement expected, regardless
Disease engages before miDnm1a fully expressed?
Dose not high enough?
Transduction inefficient?

33. David Goldstein & Michael Boland Lab
Sophie Colombo, PhD
Chris Bostick, PhD
Sarah Dugger
Daniel Krizay
Wayne Frankel Lab
Megha Sah, PhD
*JJ Teoh, PhD
*Ariadna Amador, PhD
*Sabrina Petri
*Ayla Kanber
Chana Rosenthal-Weiss
*Virginia Aimiuwu
Devin Jones
Wanqi Wang
Mouse NeuroBehavior Core
Mu Yang, PhD, Director
Elizabeth Rafikian
IGM Electrophysiology
Damian Williams, PhD
Acknowledgements
NIH R37 NS031348
NIH JCPG U54 OD020351Columbia Columbia
Univ. Precision Medicine
The Jackson Laboratory
Cat Lutz
University of Vermont
Amy Shore & Matt Weston
Emory University
Stephen Traynelis/Hongjie Yuan, Scott Myers &labs
Nationwide Children’s Hospital
Scott Harper
© Wayne N. Frankel, Ph.D.