1. HIGH CONTENT SCREENING
IDENTIFIES NOVEL REGULATORS
OF CNS AXON REGENERATION
Murray Blackmore
The Miami Project to Cure Paralysis
University of Miami

2. Why can’t axons in the CNS regenerate?

3. Why can’t axons in the CNS regenerate?
Myelin-associated inhibitors
Glial Scarring (CSPGs)
Ephrins / Semaphorins

4. Why can’t axons in the CNS regenerate?
1. Neutralize inhibitory molecules
2. Block inhibitory signaling pathways
3. Tissue / cell transplants

5. Why can’t axons in the CNS regenerate?
1. Neutralize inhibitory molecules
2. Block inhibitory signaling pathways
3. Tissue / cell transplants
≠
robust regeneration

6. Why can’t axons in the CNS regenerate?
Inadequate Cell Body
Response
1. Neutralize inhibitory molecules
2. Block inhibitory signaling pathways
3. Tissue / cell transplants
≠
robust regeneration

7. Many types of neurons regenerate
Zebrafish retina
PNS
Immature CNS

8. Many types of neurons regenerate
Zebrafish retina
?
PNS
Immature CNS

9. In vivo, rodent corticospinal tract (CST) neurons lose
regenerative capacity between postnatal day 3 and 16

10. In vivo, rodent corticospinal tract (CST) neurons lose
regenerative capacity between postnatal day 3 and 16
Research strategy
1. Comparative gene profiling
2. Functional screening in culture
3. Functional gene testing in vivo

11. What CST genes change in expression between P3
and P16?
Change in CST gene expression?
>1000 genes change 3x or more

12. Candidate genes are available in large libraries
Change in CST gene expression?
>1000 genes change 3x or more
Open Biosystems Library
6,200 human genes
9,800 mouse genes
About 160 96-well plates

13. Workflow for in vitro screening

14. Automated quantification of neurite outgrowth
~ 800 plasmids, 500 genes
1,000,000s of neurons

15. Total neurite length (normalized)
20
40
60
80
0
180
200
100
120
140
160
1500001H12RIK
4933407N01RIK
ALDOA
ATP6V1D
ALDOA
CHN1
DNAJB9 KLF4 CHN1
IDH3A
KLF10
MAT2B
PLP1
NGEF
RAB24
SCG2
SLC2A3
WBP2
SLC2A3
ALDOA
C1QDC2
CAMK2G
CREG1
DTNA
ITM2B
MKKS
OSBPL1A
RAB24
Developmentally upregulated genes
ST3GAL5
TPP1
ACTB
EZH2
SBK1
Dev.
ST8SIA4 (2)
DCL
003 SYP
DCL
EEF1G (1)
KLF6
DCX
RPL13
Screening Identifies regulators of neurite outgrowth
Genes
SET
KLF6

16. Total neurite length (normalized)
20
40
60
80
0
180
200
100
120
140
160
1500001H12RIK
4933407N01RIK
ALDOA
ATP6V1D
ALDOA
CHN1
DNAJB9 KLF4 CHN1
IDH3A
KLF10
MAT2B
PLP1
NGEF
RAB24
SCG2
SLC2A3
WBP2
SLC2A3
ALDOA
C1QDC2
CAMK2G
CREG1
DTNA
ITM2B
MKKS
OSBPL1A
RAB24
Developmentally upregulated genes
ST3GAL5
TPP1
ACTB
EZH2
SBK1
Dev.
ST8SIA4 (2)
DCL
003 SYP
DCL
EEF1G (1)
KLF6
DCX
RPL13
Screening Identifies regulators of neurite outgrowth
Genes
SET
KLF6

17. Multiple KLF family members regulate neurite
outgrowth
Neurite length (um)
KLF9
Moore, Blackmore et al. (2009). Science, 326: 298-301.

18. Full length
Domain Swap
Zinc finger only
VP16 (activation)
Engrailed (repression)
Total neurite length (um)
100
200
300
400
500
0
EGFP
*
KLF7
KLF7(ZF)
*
VP16-KLF7(ZF)
Eng-KLF7(ZF)
**
Total neurite length (um)
KLF9-KLF7(ZF)
100
200
300
400
500
0
**
Total neurite length (µm)
Total neurite length (um) (um)
Total neurite length neurite length (um)
Total
EGFP
100
200
300
400
500
0
100
100
200
200
300
300
400
400
500
500
0
0
100
200
300
400
500
0
*
KLF7
EGFP
EGFP EGFP EGFP
KLF7(ZF)
*
*
*
KLF7 KLF7
*
KLF7
*
VP16-KLF7(ZF) KLF7
KLF7(ZF) KLF7 Zinc Finger
KLF7(ZF) KLF7(ZF)
Eng-KLF7(ZF)
**
*
VP16-KLF7
*
*
*
16-KLF7(ZF)
VP16-KLF7(ZF) VP16-KLF7(ZF)
KLF9-KLF7(ZF)
Eng-KLF7
**
ng-KLF7(ZF)
Eng-KLF7(ZF) Eng-KLF7(ZF)
**
**
** **
F9-KLF7(ZF)
KLF9-KLF7(ZF) KLF9-KLF7(ZF)
**
**
**
Structure/function studies identify critical domains

19. Testing gene function in vivo
Gene delivery to Dorsal hemisection
CST cell bodies
1. Knock down growth suppressive genes
2. Over-express growth promoting genes
3. Overexpress/knock down in combination

20. AAV8 effectively transduces neurons
Thanks to:
University of Miami
Viral Vector Core

21. Viral-mediated protein knockdown in vivo
EGFP PTEN IHC Overlay

22. Viral-mediated protein knockdown in vivo
EGFP PTEN IHC Overlay
Anti-PTEN
Anti-luciferase
control

23. Control animals have minimal axon growth
below injury site
Collaboration:
Dr. Christopher Shields
Norton Neurological
Louisville, KY

24. Number of axons
100
150
200
50
0
Control
anti-NGEF
anti-PTEN
anti-SOCS3
anti-KLF2
anti-KLF5
anti-KLF15
250µm
anti-KLF9
Rheb1
CS 1
KLF7
VP16-KLF7
Number of axons
10
20
30
40
50
60
70
0
injury in vivo
Control
anti-NGEF
anti-PTEN
anti-SOCS3
anti-KLF2
anti-KLF5
anti-KLF15
anti-KLF9
1000µm
Rheb1
CS 1
KLF7
Quantification of axon growth after spinal cord
VP16-KLF7

25. Over-expression of VP16-KLF7 in CST neurons
promotes axon growth in the injured spinal cord

26. Over-expression of VP16-KLF7 in CST neurons
promotes axon growth in the injured spinal cord
**
**
**
*

27. 2. In vitro screening
3. Hypothesis driven research
4. Viral-mediated gene testing in vivo
Full length
Domain Swap
Total neurite length (normalized)
Zinc finger only
20
40
60
80
0
100
120
140
160
180
200
1500001H12RIK
4933407N01RIK
ALDOA
ATP6V1D
VP16 (activation)
CHN1
DNAJB9
IDH3A
KLF10
MAT2B
PLP1
RAB24
SCG2
SLC2A3
WBP2
ALDOA
C1QDC2
1. Cell-cell comparisons (Microarray, RNAseq)
CREG1
DTNA
Engrailed (repression)
ITM2B
MKKS
OSBPL1A
RAB24
ST3GAL5
TPP1
ACTB
EZH2
Total neurite length (um) SBK1
ST8SIA4 (2)
003 SYP
DCL
EEF1G (1)
KLF6
RPL13
SET
100
200
300
400
500
0
EGFP
*
KLF7
KLF7(ZF)
*
VP16-KLF7(ZF)
Eng-KLF7(ZF)
**
Total neurite length (um)
KLF9-KLF7(ZF)
100
200
300
400
500
0
**
Total neurite length (um) (um)
Total neurite length neurite length (um)
Total
EGFP
100
100
200
200
300
300
400
400
500
500
0
0
100
200
300
400
500
0
*
KLF7
EGFP EGFP EGFP
KLF7(ZF)
*
*
*
KLF7 KLF7 KLF7
*
VP16-KLF7(ZF)
KLF7(ZF)
KLF7(ZF) KLF7(ZF)
Eng-KLF7(ZF)
**
*
*
VP16-KLF7(ZF)
VP16-KLF7(ZF) VP16-KLF7(ZF)
KLF9-KLF7(ZF)
**
Summary: a pipeline for therapeutic gene discovery
Eng-KLF7(ZF)
Eng-KLF7(ZF) Eng-KLF7(ZF)
**
**
* **
KLF9-KLF7(ZF)
KLF9-KLF7(ZF) KLF9-KLF7(ZF)
**
**
**

28. 2. In vitro screening
3. Hypothesis driven research
4. Viral-mediated gene testing in vivo
Full length
Domain Swap
Total neurite length (normalized)
Zinc finger only
20
40
60
80
0
100
120
140
160
180
200
1500001H12RIK
4933407N01RIK
ALDOA
ATP6V1D
VP16 (activation)
CHN1
DNAJB9
IDH3A
KLF10
MAT2B
PLP1
RAB24
SCG2
SLC2A3
WBP2
ALDOA
C1QDC2
1. Cell-cell comparisons (Microarray, RNAseq)
CREG1
DTNA
Engrailed (repression)
ITM2B
MKKS
OSBPL1A
RAB24
ST3GAL5
TPP1
ACTB
EZH2
Total neurite length (um) SBK1
ST8SIA4 (2)
003 SYP
DCL
EEF1G (1)
KLF6
RPL13
SET
100
200
300
400
500
0
EGFP
*
KLF7
KLF7(ZF)
*
VP16-KLF7(ZF)
Eng-KLF7(ZF)
**
Total neurite length (um)
KLF9-KLF7(ZF)
100
200
300
400
500
0
**
Total neurite length (um) (um)
Total neurite length neurite length (um)
Total
EGFP
100
100
200
200
300
300
400
400
500
500
0
0
100
200
300
400
500
0
*
KLF7
EGFP EGFP EGFP
KLF7(ZF)
*
*
*
KLF7 KLF7 KLF7
*
VP16-KLF7(ZF)
KLF7(ZF)
KLF7(ZF) KLF7(ZF)
Eng-KLF7(ZF)
**
*
*
VP16-KLF7(ZF)
VP16-KLF7(ZF) VP16-KLF7(ZF)
KLF9-KLF7(ZF)
**
Summary: a pipeline for therapeutic gene discovery
Drug discovery
Eng-KLF7(ZF)
Eng-KLF7(ZF) Eng-KLF7(ZF)
**
**
* **
Targeted growth-
KLF9-KLF7(ZF)
KLF9-KLF7(ZF) KLF9-KLF7(ZF)
**
**
**
promotion via virus

29. Acknowledgements
Vance Lemmon Jeff Goldberg
Blackmore lab Darcie Moore
John Bixby
Zimei Wang Jessica Lerch-Haner
Yania Martinez Dario Motti
Andrea Johnstone Christopher Shields
Orisley Franch Tatiana Slepak Ping Zhang
Mathew Saccino Yan Shi
University of Miami
Viral Vector Core
National Institutes of Health (NINDS, NICHD) Anthony Oliva
The Buoniconti Fund
Craig Neilsen Foundation Pingping Jia
Ralph Wilson Foundation
Wallace Coulter Foundation
Walter G. Ross Foundation
DOD