Surrozen : SZN-043 Hepatocyte Proliferation in 14-day daily dosing Edu-labeling study

SZN-043 Induced Quick and Robust Hepatocyte Proliferation in a 14-Day Daily Dosing Edu-labeling Study in SCID Mice

Chenggang Lu*, Russell B. Fletcher, Jacqueline L. Phipps, Darshini Shah, Shalaka Deshmukh, Trevor Fisher, Hélène Baribault, Wen-Chen Yeh, Yang Li

Surrozen Inc., South San Francisco, CA 94080, USA; *Presenting author

Background

• Wnt/β-cateninsignaling plays a key role in hepatocyte regeneration in homeostasis and after liver injury
• R-spondins(RSPO) amplify the signal of Wnt ligands by stabilizing and increasing the amount of Wnt receptors on the cell surface
• We previously showed that our hepatocyte-targeted RSPO mimetic, SZN-043, specifi cally induced Wnt target gene expression in the liver and promoted hepatocyte proliferation in mice
• This study aimed to evaluate the kinetics and the overall effects of SZN-043-induced hepatocyte proliferation in a 2-week daily SZN-043 dosing schedule

Methods

• Edu was used to label any proliferating nucleus as it passes through S phase

• Liver sections were co-stained for EdU, MKI67 (proliferation marker) immunofl uorescence, and HNF4A (hepatocyte-specifi c marker)

immunofl uorescence, and counterstained with DAPI nuclear labeling

• The hepatocyte proliferation index (number of MKI67+/HNF4A+/DAPI+ nuclei) and the accumulated number of hepatocytes that had gone through at least one round of proliferation (EdU+/HNF4A+/DAPI+ nuclei) were quantifi ed

Results

Figure 2. Liver mRNA Expression of Axin2 was Significantly Increased with SZN-043

	0.15		Liver Axin2 expression		• Axin2 is a Wnt/β-catenin
					Anti-GFP	target gene and an indicator of
		*				SZN-043 activity in the liver.
					SZN-043
			*		• Liver mRNA expression
Fold/Gapdh					*		of Axin2 was signifi cantly
	*			**	increased with SZN-043
0.10	*	**		treatment compared to
	*			anti-GFP throughout the study,
							indicating that continuous
		**					dosing of SZN-043 maintained
						β-catenin signaling induction
Relative							despite the low level of
0.05						hepatocyte proliferation after
							day 5.
							• This result together with
							Figures 1A and 1B suggested
							hepatocytes only go through
	0.00						a limited number of cell cycles
	0	5		10	15	when continuously exposed to
		Day	SZN-043 in the 14-day study.

Mean with SD. Statistical Analyses: Two-way ANOVA, with Holm-Šídák correction.

All comparisons made with the anti GFP group. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

Figure 3. Non-Hepatocyte(HNF4A-negative) Cell Proliferation was Significant with SZN-043

(C)

					• There was negligible
cadherin-					proliferation of
				cholangiocytes (epithelial
				cells of the bile duct; labeled
					with CK19) with SZN-043
					(Figure 3D).
VE					• As previous studies with
				SZN-043 have shown no
					direct impact on cells other
					than hepatocytes (data
					on fi le), the proliferation
(D)	Day 1	Day 4	Day 7	Day 14	observed in the HNF4A-
negative cells in this study
					was likely a secondary
GFP					effect in response to and in
				support of the increasing
				number of hepatocytes.
Anti-

SZN-043

Figure 1. Significant Proliferation of Hepatocytes in Response to SZN-043 Was Quick and Transient

(A)

Ki67+Hnf4a+EdU+Nuclei

• Signifi cant hepatocyte

2500

proliferation was observed

48 hours after the fi rst dose

2000

Anti-GFP

of SZN-043, reaching a peak

SZN-043

at Day 4, followed by a rapid

Nuclei

1000

decline to near baseline

900

***

level at Day 6 (Figure 1A).

800

Consistently, the percent of

of

700

**

hepatocyte nuclei that were

600

EdU positive reached a

Number

500

plateau at Day 5 (Figure 1B).

400

These together suggest that

300

**

most hepatocytes stopped

200

proliferating or incorporating

100

**

**

EdU after this timepoint.

0

• These fi ndings suggest that

1

2

3

4

5

6

7

8

9

10

11

12

13

14

the hepatocytes responded

Day

quickly and robustly to

SZN-043, but only underwent

(B)

Percent of HNF4a+ Nuclei that are EdU+

a limited number of

proliferation cycles, despite

80

the continued exposure to

****

***

****

****

SZN-043 for up to 14 days.

****

• Over 50% of all hepatocytes

60

**** ****

in the SZN-043-treated group

Percent

****

were EDU+ by study end

40

compared to about 10% in

***

the anti-GFP-treated group

(Figure 1B), demonstrating

20

the pharmacology effect

**

of SZN-043 on stimulating

hepatocyte regeneration.

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Day

(A)

(B)

Intensity sum

4 × 108

F4/80

Anti-GFP

3 × 108

****

SZN-043

****

2 × 108

*

** *

1 × 108

0

0	5	Day	10	15

Mean with SD. Statistical Analyses: One-way ANOVA, Holm-Šídák test. All comparisons made with the anti GFP group.

*p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

• Signifi cant non-hepatocyte(HNF4A-negative) cell proliferation was also observed with SZN-043
• • Proliferation of non- hepatocyte appeared to have peaked after hepatocyte proliferation
  • Mostly of Kupffer cells/ macrophages
  • • F4/80 (macrophage and monocyte marker; red) expression peaked signifi cantly at Day 5 compared to anti-GFP (Figure A and B). (liver sections counted stained with DAPI; white)
    • After Day 8, F4/80 expression was comparable between SZN-043 and anti- GFP, with no visible differences observed.
  • Endothelial cell marker staining did not reveal differences between SZN-043 and anti-GFP groups (Figure 3C).

Conclusions

• SZN-043induced a rapid transient increase in hepatocyte proliferation, followed by a rapid decline to baseline despite continued exposure to SZN-043 and β-catenin signaling induction for up to 14 days.
• Hepatocyte proliferation induced by SZN-043 was self-limited and likely controlled by other mechanisms controlling cell proliferation (Sarkar A. et al., 2021).
• Proliferation observed in the HNF4A-negative cells was likely due to the tissue responding to the large number of proliferating hepatocytes.

Presenting author: Chenggang Lu, email: [e-mail]

Surrozen Inc., South San Francisco, CA 94080 - www.surrozen.com

Previously presented at Digestive Disease Week (DDW), San Diego, CA, May 21-24, 2022

CL, RBF, JLP, DS, SD, TF, HB, WY and YL are present or past employees of Surrozen, Inc. and shareholders of Surrozen stock.

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