Emily Buck Whitaker Conference April 30, 2015 Engineering the growth factor, CXCL12 α, for heart tissue regeneration

Cardiovascular diseases account for the highest number of deaths worldwide 1. 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,

What is damaged by reduced blood supply? -3-

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: , M. Zhang et al, The FASEB Journal, 21: , 2007 CXCL12α promotes 2 … 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 apoptosis 3 Promote rebuilding of capillaries and small arterioles 3 PROBLEM: Short half-life and easy diffusion! 2 -4-

Previous studies in Dr. Hubbell’s lab 4 … (1)Identification of a domain of platelet-derived growth factor 2 (PlGF ) 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: , 2014 How can we improve the regenerative capacity of CXCL12α? -5-

My lab in Lausanne Dr. Jeffrey Hubbell Priscilla Briquez Laboratory for Regenerative Medicine and Pharmacobiology (LMRP) -6-

1. Design and Clone 2. Produce and Purify 3. Characterize ECM-binding affinity and bioactivity 5. D. Czajkowsky et al, EMBO Mol Med, 4: , How will we engineer CXCL12α? DESIGN: Establish sequence with the PlGF-2 domain fused to N- OR C-terminus of CXCL12 α AND Fc tag to produce wild-type 5 CLONE: PCR, agarose gel electrophoresis Restriction, ligation and transformation into plasmid Sequence verification Amplify plasmid for production PRODUCE: Transient gene expression of CXCL12 α variants in mammalian cells SDS-Page, Western blotting PURIFY: Separate CXCL12 α from contaminants through His-tag affinity chromatography Size exclusion chromatography CHARACTERIZE: Binding affinity for ECM components through… ELISA Release from fibrin hydrogels Bioactivity testing through… MSC migration assay Phosphorylation assay -7-

Ligate in plasmid by restriction enzymes I. DESIGN AND CLONE KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK wtCXCL12α 68 AA SHL-CXCL12α 90 AA CXCL12α-SHL 90 AA RRRPKGRGKRRREKQRPTDSHLKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLK NNNRQVCIDPKLKWIQEYLEKALNK KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN KRRRPKGRGKRRREKQRPTDSHL PlGF AA RRRPKGRGKRRREKQRPTDCHL CXCL12α-SHL (288 bp) SHL-CXCL12α (288 bp) CXCL12-SHL OR SHL-CXCL12 PLASMID: pXLG*Chis AgeI BamHI Transform into DH5α competent bacteria Verify DNA sequence -8-

Ligate in plasmid by restriction enzymes I. DESIGN AND CLONE KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK wtCXCL12α 68 AA RRRPKGRGKRRREKQRPTDSHLKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLK NNNRQVCIDPKLKWIQEYLEKALNK PlGF AA RRRPKGRGKRRREKQRPTDCHL CXCL12-SHL OR SHL-CXCL12 PLASMID: pXLG*Chis AgeI BamHI Transform into DH5α competent bacteria Verify DNA sequence CXCL12α-SHL (288 bp) SHL-CXCL12α (288 bp) SHL-CXCL12α 90 AA CXCL12α-SHL 90 AA -9- KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN KRRRPKGRGKRRREKQRPTDSHL

Ligate in plasmid by restriction enzymes I. DESIGN AND CLONE KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK wtCXCL12α 68 AA Fc domain AA Transform into DH5α competent bacteria DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK + FLAG cleavage site CXCL12-Fc PLASMID: pXLG*Chis AgeI HindIII CXCL12α (204 bp) Fc Tag (786 bp) Ligate two fragments by restriction enzymes CXCL12 α -Fc (1056 bp) Amplify band of correct size by PCR CXCL12 α -Fc (984 bp) Verify DNA sequence 6. J.W. Murphy et al, J Biol Chem, 282: ,

50 kDa 15 kDa 10 kDa II. PRODUCE AND PURIFY HEK ± VPA CHO ± DMSO wt CXCL12α 9.2 kDa 1.Supernatant + 2.Supernatant – 3.PBS wash + 4.PBS wash – 1.NaCl wash + 2.NaCl wash – 3.Pellet + 4.Pellet - HEK CHO CXCL12α-Fc 36.6 kDa CXCL12α-SHL 12.1 kDa SHL-CXCL12α 12.1 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 PURIFY Fc 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 CXCL12α-SHL-Fc (39 kDa) 2.SHL-CXCL12α-Fc (39 kDa) 50 kDa 15 kDa 10 kDa 11* 22* 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 Elution buffer Protein in elution buffer As-received Day 7 supernatant -13-

* Enterokinase (%) per weight CXCL12α-Fc % % (1 unit) % II. PRODUCE AND PURIFY Cleave Fc tag from CXCL12α-Fc % % 0.01% CXCL12α Fc Tag FLAG + Enterokinase (31 kDa) CXCL12α + Fc Tag FLAG 37 kDa 9 kDa 28 kDa Incubate RT Incubation time at room temperature 1.4h 2.8h 3.16h VARIABLE 1:VARIABLE 2: EK to CXCL12α-Fc -14-

3. Western blot anti-his 1 1* 2 2* 3 4 4* 5 5* 6 6* *13* 14* * Size exclusion chromatography To separate cleaved Fc tag from CXCL12α II. PRODUCE AND PURIFY Separate Fc tag from CXCL12α Fc tag + EK CXCL12α Western blot To confirm presence of CXCL12α after cleavage 1.Before his purification 2.After his purification 3.Dialysis in 1X PBS 4.After EK cleavage 1.Size exclusion Size exclusion Size exclusion

NEXT STEPS… II. PRODUCE AND PURIFY 1. CXCL12α-SHL-Fc Optimize cleavage of Fc tag from CXCL12α-SHL Separate CXCL12α-SHL from tag and enzymes Perform western blot to confirm presence of CXCL12α 2. CXCL12α Mass spectrometry and amino acid sequencing 3. SHL-CXCL12α-Fc Optimize production III. CHARACTERIZE Binding to ECM components Study bioactivity of CXCL12α Compare binding and bioactivity of CXCL12α-SHL with that of CXCL12γ -16-

-17- 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 staining Immunohistochemistry Flow cytometry

Acknowledgements Dr. Jeffrey Hubbell Priscilla BriquezJean-Phillipe Gaudry Protein Expression Core Facility Fellow members of LMRP -18- ERC Cytrix Grant

Thank you for your attention QUESTIONS? -19-

S1. Previous studies by LMRP GF binding to ECM proteins, measured by ELISA 4 4. M. Martino et al, Science, 343: , 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 -20-

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 -21-

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 -22-

S4. Restriction of CXCL12α variants 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) 1.CXCL12-SHL (BamHI &BgIII) 2.CXCL12-Fc (BamHI &BgIII) 3.SHL-CXCL12 (BamHI &BgIII) 4.CXCL12-Fc (BamHI &BgIII)

S5. SDS-page for western blots 3. Western blot anti-his 1 1* 2 2* 3 4 4* 5 5* 6 6* SDS-page staining of gel after WB transfer Western blot with anti-CXCL12 1 1* 2 2* 3 4 4* 5 5* 6 6* Before his purification 2.After his purification 3.Dialysis in 1X PBS 4.After EK cleavage 1.Size exclusion Size exclusion Size exclusion