Emily Buck

Whitaker Conference

April 30, 2015

Engineering the growth factor, CXCL12α, for heart tissue regeneration

Cardiovascular diseases account for the highest number of deaths worldwide1.

MAJOR CONSEQUENCES:

Loss in cardiac function

Possibility of complications

Heart tissue is damaged when blood supply is totally cut off from heart tissue

HEART ATTACK (MYOCARDIAL INFARCTION)

Blockage of coronary arteries causes a reduction in blood supply to heart tissue

CORONARY (ISCHEMIC) HEART DISEASE

1. World Health Organization, “Global status report on noncommunicable diseases,” p. 9, 2011 -2-

What is damaged by reduced blood supply?

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Ischemic tissue transiently expresses growth factors, such as VEGF and CXCL12α (SDF-1α), to initiate mechanisms for revascularization in the damaged area

2. C. Cencioni et al, Cardiovascular Research, 94: 400-407, 20123. M. Zhang et al, The FASEB Journal, 21: 3197-3207, 2007

CXCL12α promotes2…• expression of its

receptor, CXCR4• migration of progenitor

and endothelial cells to ischemic area

After myocardial infarction, overexpression of CXCL12α has been shown to

• Reduce cardiac myocyte apoptosis3

• Promote rebuilding of capillaries and small arterioles3

PROBLEM: Short half-life and easy diffusion!

2

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Previous studies in Dr. Hubbell’s lab4…

(1) Identification of a domain of platelet-derived growth factor 2 (PlGF-2123-144) binds with high affinity to many ECM proteins

(2) Fusion of this PlGF-2 domain to growth factors (BMP-2, PDGF-BB, and VEGF-A) enhanced their affinities for the ECM

(3) Engineering the growth factors by fusion of the PlGF-2 domain improved their capacity for wound healing and lowered the dose required for efficacy

Let’s try with CXCL-12α!

4. M. Martino et al, Science, 343: 885-888, 2014

How can we improve the regenerative capacity of CXCL12α?

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My lab in Lausanne

Dr. Jeffrey Hubbell

Priscilla Briquez

Laboratory for Regenerative Medicine and Pharmacobiology (LMRP)

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1. Design and Clone 2. Produce and Purify

3. Characterize ECM-binding affinity and bioactivity

5. D. Czajkowsky et al, EMBO Mol Med, 4: 1015-1028, 2012.

How will we engineer CXCL12α?

DESIGN:Establish sequence with the

PlGF-2 domain fused toN- OR C-terminus of CXCL12α

AND Fc tag to produce wild-type5

CLONE:PCR, agarose gel electrophoresis

Restriction, ligation and transformation into plasmid

Sequence verificationAmplify plasmid for production

PRODUCE:Transient gene expression of

CXCL12α variants in mammalian cells

SDS-Page, Western blotting

PURIFY:Separate CXCL12α from

contaminants through

His-tag affinity chromatography

Size exclusion chromatography

CHARACTERIZE:

Binding affinity for ECM components through…

ELISARelease from fibrin hydrogels

Bioactivity testing through…

MSC migration assay

Phosphorylation assay

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Ligate

in plasmid by restrictionenzymes

I. DESIGN AND CLONEKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKwtCXCL12α

68 AA

SHL-CXCL12α90 AA

CXCL12α-SHL90 AA

RRRPKGRGKRRREKQRPTDSHLKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKRRRPKGRGKRRREKQRPTDSHL

PlGF-2123-14422 AA

RRRPKGRGKRRREKQRPTDCHL

CXCL12α-SHL (288 bp)

SHL-CXCL12α(288 bp) CXCL12-SHL

ORSHL-CXCL12

PLASMID:pXLG*Chis

AgeI

BamHI

200300

200300

Transforminto DH5α competent

bacteria

Verify DNA sequence

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Ligate

in plasmid by restrictionenzymes

I. DESIGN AND CLONEKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKwtCXCL12α

68 AA

RRRPKGRGKRRREKQRPTDSHLKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

PlGF-2123-14422 AA

RRRPKGRGKRRREKQRPTDCHL

CXCL12-SHL OR

SHL-CXCL12

PLASMID:pXLG*Chis

AgeI

BamHI

Transforminto DH5α competent

bacteria

200300

200300

Verify DNA sequence

CXCL12α-SHL (288 bp)

SHL-CXCL12α(288 bp)

SHL-CXCL12α90 AA

CXCL12α-SHL90 AA

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KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKRRRPKGRGKRRREKQRPTDSHL

Ligate

in plasmid by restrictionenzymes

I. DESIGN AND CLONEKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKwtCXCL12α

68 AA

Fc domain6

227 AA

Transform

into DH5α competent

bacteria

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK + FLAG cleavage site

CX

CL12-FcPLASMID:

pXLG*Chis

AgeI

HindIII

CXCL12α (204 bp)

200

300

Fc Tag (786 bp)

700

800 Ligate

two fragmentsby restrictionenzymes

CXCL12α-Fc (1056 bp)

Amplify

band of correct size

by PCR

CXCL12α-Fc (984 bp)

Verify DNA sequence

6. J.W. Murphy et al, J Biol Chem, 282: 10018-10027, 2007 -10-

50 kDa 15 kDa 10 kDa

II. PRODUCE AND PURIFYHEK ± VPA CHO ± DMSO

wt CXCL12α9.2 kDa

1. Supernatant +2. Supernatant –3. PBS wash +4. PBS wash –

5. NaCl wash +

6. NaCl wash –7. Pellet +8. Pellet -

HEK

CHO

CXCL12α-Fc36.6 kDa

CXCL12α-SHL12.1 kDa

SHL-CXCL12α12.1 kDa

SDS-page for Day 7 supernatant, PBS wash, NaCl wash, and pellet

Need to add Fc for PlGF-2 variants-11-

II. PRODUCE AND PURIFYFc tag for PlGF-2 variants

CXCL12-SHL

OR

SHL-CXCL12

PLASMID:pXLG*Chis Fc

Tag

BamHI

BgI II

Ligate Fc tag in PlGF-2 variant plasmids with restriction

enzymes

Transform into DH5α competent

bacteria

Verify DNA sequence

AgeI

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1. CXCL12α-SHL-Fc (39 kDa)2. SHL-CXCL12α-Fc (39 kDa)

50 kDa 15 kDa 10 kDa

1 1* 2 2*

HEK + VPA

II. PRODUCE AND PURIFY His-affinity for CXCL12α-Fc

His purification fractions(left to right) A6, A6*, A7, A11, A13, B15, B15*, B13, B11, B5, B3, B1, B1*, and C3

Supernatant with CXCL12α-Fc

Contaminants

1. 2.

1. 2.

Elution buffer

Protein in elution buffer

As-received Day 7

supernatant

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4 8 16 4 8 16* 4 8 16 0

50

25

10

Enterokinase (%) per weight CXCL12α-Fc

1. 0.0001% 2. 0.00064% (1 unit)

3. 0.01%

II. PRODUCE AND PURIFY Cleave Fc tag from CXCL12α-Fc 0.0001% 0.00064% 0.01%

CXCL12α Fc TagFLAG

+ Enterokinase (31 kDa)

CXCL12α + Fc TagFLAG

37 kDa

9 kDa 28 kDa

Incubate RT

Incubation time at room temperature

1. 4h2. 8h3. 16h

VARIABLE 1: VARIABLE 2:

EK to CXCL12α-Fc

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3. Western blot anti-his

1 1* 2 2* 3 4 4* 5 5* 6 6* 7 8

10

25

50

12 12* 13* 14*14 29 30 30* 31 32 33 34 35 36

Size exclusion chromatographyTo separate cleaved Fc tag from CXCL12α

II. PRODUCE AND PURIFY Separate Fc tag from CXCL12α

Fc tag + EK CXCL12α

Western blotTo confirm presence of CXCL12α after cleavage

1. Before his purification2. After his purification3. Dialysis in 1X PBS4. After EK cleavage

5. Size exclusion 12-16

6. Size exclusion 29-317. Size exclusion 32-35

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NEXT STEPS…II. PRODUCE AND PURIFY 1. CXCL12α-SHL-Fc

Optimize cleavage of Fc tag from CXCL12α-SHLSeparate CXCL12α-SHL from tag and enzymesPerform western blot to confirm presence of

CXCL12α

2. CXCL12αMass spectrometry and amino acid sequencing

3. SHL-CXCL12α-FcOptimize production

III. CHARACTERIZE

Binding to ECM componentsStudy bioactivity of CXCL12αCompare binding and bioactivity of CXCL12α-

SHL with that of CXCL12γ -16-

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In-vivo model

1. Induce infarction by coronary artery ligation and reperfuse

2. Inject saline, wtCXCL12α, wtCXCL12γ, OR CXCL12α-PlGF2 variant into infarcted area after surgery

3. Study regeneration of myocardium through

H&E stainingImmunohistochemistryFlow cytometry

Acknowledgements

Dr. Jeffrey Hubbell Priscilla Briquez Jean-Phillipe Gaudry

Protein Expression Core Facility

Fellow members of LMRP

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ERC Cytrix Grant

Thank you for your attention

QUESTIONS?

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S1. Previous studies by LMRP

GF binding to ECM proteins, measured by ELISA4

4. M. Martino et al, Science, 343: 885-888, 2014

Representative histology at 15 days for the topical groups (hematoxylin and eosin staining). Black

arrows indicate wound edges; red arrows indicate tips of the healing epithelium tongue. Scale bar, 1

mm. 4

Db/db mice with skin wound

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AAG CCC GTC AGC CTG AGC TAC AGA TGC CCA TGC CGA TTC TTC GAA AGC CAT GTT GCC AGA GCC AAC GTC AAG CAT CTC AAA ATT CTC AAC ACT CCA AAC TGT GCC CTT CAG ATT GTA GCC CGG CTG AAG AAC AAC AAC AGA CAA GTG TGC ATT GAC CCG AAG CTA AAG TGG ATT CAG GAG TAC CTG GAG AAA GCT TTA AAC AAG CGC AGA CGA CCG AAA GGT AGA GGC AAG AGG AGA CGA GAG AAG CAG AGG CCG ACC GAT AGT CAT CTG

CXCL12-SHL

1. CXCL12 –SHL Rev1 (57 bp)5’-CGTCTCCTCTTGCCTCTACCTTTCGGTCGTCTGCGCTTGTTTAAAGCTTTCTCCAGG-3’ 2. Rev2 SHL+R (66 bp)5’- ATAATATGGATCCGCGCAGATGACTATCGGTCGGCCTCTGCTTCTCTCGTCTCCTCTTGCCTCTAC-3’

1-2. MCS CXCL12 Fwd (40 bp)5’-AATTACCGGTGAC AAGCCCGTCAGCCTGAGCTACAGATGC-3’

CGC AGA CGA CCG AAA GGT AGA GGC AAG AGG AGA CGA GAG AAG CAG AGG CCG ACC GAT AGT CAT CTG AAG CCC GTC AGC CTG AGC TAC AGA TGC CCA TGC CGA TTC TTC GAA AGC CAT GTT GCC AGA GCC AAC GTC AAG CAT CTC AAA ATT CTC AAC ACT CCA AAC TGT GCC CTT CAG ATT GTA GCC CGG CTG AAG AAC AAC AAC AGA CAA GTG TGC ATT GAC CCG AAG CTA AAG TGG ATT CAG GAG TAC CTG GAG AAA GCT TTA AAC AAG

SHL-CXCL12

1. SHL-CXCL12 Fwd (50 bp) 5’-CAGAGGCCGACCGATTGTCATCTGAAGCCCGTCAGCCTGAGCTACAGATG-3’ 2. Fwd A - SHLdom (55 bp)5’- GAAAGGTAGAGGCAAGAGGAGACGAGAGAAGCAGAGGCCGACCGATAGTCATCTG-3’

3. Fwd B – SHLdom (58 bp)5’- TATATTACCGGTGACCGCAGACGACCGAAAGGTAGAGGCAAGAGGAGACGAGAGAGGC-3’

1-3. MCS CXCL12 Rev (50 bp)5’-GCCTGCTGGATCC CTTGTTTAAAGCTTTCTCCAGGTACTCCTGAATCCAC-3’

S2. Primers

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S3. Methods to ligate CXCL12α and Fc tag

1. CXCL12α + Fc then add plasmid vector in one reaction after 1hr of incubation

1. Varied ratio of CXCL12 to Fc

2. CXCL12α + Fc with two restriction sites

3. CXCL12α + Fc with one restriction site

4. Cut band from ligation of only CXCL12α and Fc at correct size and amplify by PCR

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S4. Restriction of CXCL12α variants5

8

741 2 3

1. CXCL12-SHL (AgeI & BamHI)2. CXCL12noHindIII (BamHI)3. Fc tag (BamHI)4. Fc tag (BamHI &HindIII)5. pXLG (BamHI & AgeI)6. pXLG (AgeI & HindIII)7. CXCL12-Fc (AgeI & HindIII)

8. CXCL12-SHL (BamHI &BgIII)9. CXCL12-Fc (BamHI &BgIII)10. SHL-CXCL12 (BamHI &BgIII)11. CXCL12-Fc (BamHI &BgIII)

6

9 10 11

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S5. SDS-page for western blots

3. Western blot anti-his

1 1* 2 2* 3 4 4* 5 5* 6 6* 7 8

10

25

50

SDS-page staining of gel after WB transfer Western blot with anti-CXCL12

1 1* 2 2* 3 4 4* 5 5* 6 6* 7 8

1. Before his purification2. After his purification3. Dialysis in 1X PBS4. After EK cleavage

5. Size exclusion 12-16

6. Size exclusion 29-317. Size exclusion 32-35

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