Experimental Report

Experiment Number: PHER-YKYY015-07

In Vitro Efficacy Evaluation Report of YKYY015 Murine-derived Molecule

Experimental Institution:

Beijing Yuetkang Kechuang Pharmaceutical Technology Co., Ltd.

Building 3, No. 11 Kechuang 7th Street, Beijing Economic-Technological Development Area

Issuing Department: Quality Management Department QA

Table of Contents

_Toc199934527

1. Research Objective 5

2. Experimental Design 5

3. Materials 5

3.1 Test Compounds 5

3.2 Primary Cells 6

3.3 Instruments 6

3.4 Main Reagents and Consumables 7

4. Experimental Methods 8

4.1 Compound Dilution 8

4.2 Cell Digestion and Counting 8

4.3 Cell Transfection 9

4.4 RNA Extraction 9

4.5 Reverse Transcription and qPCR 9

4.6 ELISA Experiment 10

4.6.1 Sample Preparation 10

4.6.2 Experimental Method 10

4.7 Data Analysis 11

5. Experimental Results 12

6. Change History 23

Appendix 24

Table of Main Symbols and Abbreviations

Abstract

Objective:

The purpose of this study is to screen mouse-derived substitute molecules with sequences similar to YKYY015 at the level of primary rat hepatocytes (PRH), and to determine the saturating dose for inhibition of the target gene, thereby providing candidate test substances for in vivo pharmacodynamic experiments and reproductive toxicity-related studies in rats.

Methods:

A total of 13 YKYY029 mouse-derived substitute molecule sequences were diluted with sterile, enzyme-free water to 20 μM. For the formal experiment, the compound solutions (initial concentration 200 nM) were further diluted with culture medium to 8 appropriate concentration gradients, with a 4-fold difference between adjacent gradients. After 48 hours of free cellular uptake of the siRNA sequences, the cell supernatant was collected and cellular RNA was extracted. mRNA and protein expression levels were detected by qPCR and ELISA, respectively. Eight concentration points were selected for dose-response relationship experiments, and the final IC ₅₀ value was determined. Three candidate compounds were selected for in vivo pharmacodynamic validation experiments in rats.

Results:

According to the data summary, D82-DV29PG5, D82RAT1-DV29PG5, Inclisiran, sh1-DV29PG5, and PCS-A2-DV29PG5L could not have their IC50 fitted due to a low upper limit on the fitting curve. The top five sequences in the overall ranking are D82RAT6-DV29PG5, D82RAT5-DV29PG5, D82RAT3-DV29PG5/D82RAT4-DV29PG5, D82RAT2-DV29PG5, and D82RAT7-DV29PG5.

Research Objective

YKYY015 injection is an siRNA drug targeting PCSK9, currently at the NDA stage. According to relevant guidelines and based on reproductive toxicity study cases of marketed siRNA drugs, it is necessary to include research on YKYY015 injection substitute molecules in the reproductive toxicity test in rats. Therefore, the purpose of this study is to screen mouse-derived substitute molecules with sequences similar to YKYY015 at the primary rat hepatocyte (PRH) level, and to determine their saturating dose for target gene inhibition, thereby providing candidate test articles for in vivo pharmacodynamic experiments and reproductive toxicity-related studies in rats.

According to literature reports and sequence homology, 11 mouse-derived surrogate molecules with sequences similar to YKYY015 were designed. Both qPCR and ELISA methods were used to screen and evaluate the 11 surrogate molecules, YKYY015 injection, and nclisiran (names and related information are shown in Table 2), to assess their efficiency in inhibiting PCSK9 mRNA and protein expression in primary rat hepatocytes. This guided the in vivo pharmacodynamic evaluation in rats, as well as related reproductive toxicity studies and screening work.

Experimental Design

After 48 hours of free cellular uptake of siRNA sequences, cell supernatants were collected and cellular RNA was extracted. qPCR and ELISA were used to detect mRNA and protein expression levels, respectively. Eight concentration points were selected for dose-response experiments, and the final IC ₅₀ value was determined. Three candidate compounds were selected for in vivo pharmacodynamic validation experiments in rats. (For details, see "YKYY015 Mouse-Derived Molecule In Vitro Pharmacodynamic Screening Experimental Protocol" PHEP-YKYY015-07).

Materials

Test Compounds

The screening included a total of 13 compounds: alternative molecular candidate compounds designed against the rat PCSK9 mRNA sequence, the clinical molecular compound YKYY015 (No. 1), and the positive control Inclisiran (No. 9). The numbers and names of all compounds are shown in Table 2.

Table 2 Compound Information

Primary cells

Table 3 Cell Information

Instruments

The instruments used in this experiment are as follows.

Table 4 Experimental Equipment Information

Main reagents and consumables

The reagents and consumables used in this experiment are as follows.

Table 5 Reagents, Consumables, and Manufacturers

Gapdh and Pcsk9 primers (using the primer sequences confirmed during the screening phase) were synthesized by BGI. The primer sequences are shown in the table below.

Table 6 qPCR Primers

Experimental Methods

Compound Dilution

Thirteen compounds were diluted to 20 μM using sterile, enzyme-free water. For the formal experiment, the compound solutions (initial concentration of 200 nM) were further diluted with culture medium to create eight appropriate concentration gradients, with a 4-fold difference between adjacent gradients (Table 7).

Table 7 Formal Experiment Compound Concentrations

Cell digestion and counting

Remove the primary rat hepatocytes from liquid nitrogen and immediately place them in a 37°C water bath. Gently shake the cryovial for about 1 min 45 s, then quickly pour all the liquid from the cryovial into a pre-warmed 40 mL resuscitation medium. Tighten the centrifuge tube cap and gently invert the tube twice to mix the cell suspension. Centrifuge at room temperature at 50 g for 2 min. Carefully remove the supernatant with a pipette, leaving about 0.5 mL of supernatant, and add 2 mL of maintenance medium to bring the total volume to 3 mL. Take 10 μl of the cell suspension and add 10 μl of 0.4% trypan blue solution. Mix thoroughly with a pipette, then add the mixture to the counting chamber of a cell counting plate. Insert the cell counting plate into the counting slot of the Countstar BioTech cell counter and use the software to complete cell counting. After counting, dilute the suspension with plating medium according to the counting results to adjust the cell density to 2.8×10^5/mL. Gently mix the cell suspension, use a multichannel pipette to seed 100 μl of the cell suspension into a 96-well collagen-coated culture plate, and mix the cells in the plate using the "cross method."

Cell transfection

For primary rat hepatocytes, prepare 75 μl of compound/medium mixture per well, diluted proportionally. Add 50 μl of the above mixture to each well of a 96-well cell culture plate, resulting in a final volume of 150 μl per well. After plating, shake and gently tap the culture plate to mix, then incubate in a 5% CO₂, 37°C incubator for 48 hours.

RNA Extraction

After 48 hours of transfection, collect the culture medium and transfer it to a 1.5 mL centrifuge tube for measurement of PCSK9 protein expression. Extract total cellular RNA using the RNeasy kit, following these steps:

Add 500 μl PBS to wash the cells.

Aspirate PBS and add 100 μL Buffer RLT.

Add 100 μL of 70% ethanol to each well to precipitate RNA.

Mix the liquid in each well by pipetting up and down three times with a multichannel pipette, then transfer the solution containing RNA precipitate from each well to an RNeasy spin column with a collection tube.

Centrifuge at 10,000g for 1 min and discard the flow-through. Place the RNeasy spin column back into the 2 mL Collection Tube.

Add 700 μl Buffer RW1 to the RNeasy spin column, centrifuge at 10,000 g for 30 seconds, and discard the flow-through.

Add 500 μl Buffer RPE (add 4 volumes of 100% ethanol to the Buffer RPE bottle before first use) to the RNeasy spin column, centrifuge at 10,000 g for 30 seconds, and discard the flow-through.

Repeat step 7.

Place the RNeasy spin column back into a 2 mL collection tube and centrifuge at 10,000 g for 2 minutes.

Place the RNeasy spin column onto a 1.5 mL RNase-Free Collection Tube, add 30 μl of sterile, nuclease-free water to the center of the RNeasy spin column membrane, and let it stand at room temperature for 5 minutes.

Centrifuge at 10,000 g for 1 minute to elute the RNA.

Reverse Transcription and qPCR

Perform reverse transcription according to the FastKing RT Kit (with gDNase) instructions. The procedure is as follows:

gDNA removal: Add 2 μl 5×gDNA Buffer and 8 μl RNA solution, mix well, briefly centrifuge, incubate at 42°C for 3 minutes, then place on ice for later use.

Reverse transcription: Prepare the reverse transcription system mixture according to Table 8. Mix 10 μl of the reverse transcription system mixture with 10 μl of the gDNA-removed reaction solution, incubate at 42°C for 15 minutes, then at 95°C for 3 minutes, and place on ice for later use or store at low temperature.

Table 8 Reverse Transcription System

qPCR was performed using the TB Green® Premix Ex Taq™ (Tli RNaseH Plus) kit. A 10 μL reaction system was prepared in a 96-well plate as shown in the table, and qPCR was used to detect the RNA expression levels of GAPDH and PCSK9, with GAPDH serving as the reference gene. Each qPCR assay was performed in triplicate. The qPCR cycling program was as follows: 95°C for 30 s, then 40 cycles of 95°C for 10 s and 60°C for 34 s.

Table 9 qPCR Reaction System

ELISA experiment

Sample Preparation

Collection of Culture Supernatant: Collect the culture supernatant from each sample in the 96-well plate into a 96-well deep-well plate and store at -20°C. Before the formal experiment, thaw the frozen samples at room temperature and mix by vortexing.

Experimental Methods

According to the instructions of the Rat PCSK9 ELISA Kit, the operating steps are as follows:

(1) Collect the cell supernatant and either test immediately or store at -20°C. Before testing, remove the kit from the refrigerator and allow it to equilibrate at room temperature for at least 30 minutes.

(2) Dilute the wash buffer and dilution buffer from the kit 20-fold with deionized water, then mix well before use.

(3) According to the instructions on the standard label, add the appropriate volume of 1× dilution buffer to dissolve and prepare a stock solution of the standard at a concentration of 1500 pg/ml. Use 500 µl of 1× dilution buffer as the diluent, and perform six consecutive 2-fold serial dilutions of the 1500 pg/ml highest concentration standard in six separate EP tubes, preparing standards of various concentrations as shown in the table below. Note that standards should be freshly prepared before use.

Table 10 Preparation of ELISA Standard Curve

Add 100 μl of standard (for the standard curve) and 100 μl of the diluted sample to be tested into the corresponding wells of the 96-well plate pre-coated with antibody. Each sample is tested in duplicate wells. Cover the plate and incubate at room temperature for 2 hours. Discard the reaction solution, add 300 μl of 1× wash buffer to each well, and repeat washing 3 times, ensuring that the wash buffer remains in the wells for at least 2 minutes each time. Completely remove the liquid from the wells after each wash. After the final wash, invert the plate and gently tap it on a paper towel to remove excess liquid. Secondary antibody incubation: Prepare the detection antibody working solution (diluted 1:400 with 1× dilution buffer) within 10 minutes before incubation. Add 100 μl of detection antibody working solution to each well, mix gently, and incubate at room temperature for 1 hour. Discard the reaction solution, add 300 μl of 1× wash buffer to each well, and repeat washing 3 times, ensuring that the wash buffer remains in the wells for at least 2 minutes each time. Completely remove the liquid from the wells after each wash. After the final wash, invert the plate and gently tap it on a paper towel to remove excess liquid. Substrate incubation: Within 10 minutes before incubation, mix equal volumes of Substrate Solution A and Substrate Solution B, and add 100 μl of the mixed solution to each well. Incubate at room temperature in the dark for 20 minutes. Add 100 μl of stop solution to each well, mix by shaking in the dark, and read the plate within 10 minutes. Record the OD value at 450 nm.

Data analysis

For the ELISA results, the standard curve was first plotted using the four-parameter fitting function (Nonlinear regression (curve fit): [Agonist] vs response – variable slope four parameters) in GraphPad Prism 10.4.0 software, with the x-axis representing the standard concentration and the y-axis representing absorbance at 450 nm. The absorbance of the test samples was entered into the function to obtain the corresponding protein concentration, which was then multiplied by the dilution factor of the cell culture medium to determine the actual PCSK9 content in the sample. Inhibition rate (%) = (mean expression level of control group - mean expression level of sample) / mean expression level of control group × 100%.

The dose-response curve of the compound was fitted using GraphPad Prism 10.4.0 software (Nonlinear regression (curve fit): [Inhibitor] vs response – variable slope four parameters or [Agonist] vs response – variable slope four parameters), and the IC ₅₀ of the compound was calculated. All results are reported to five decimal places.

For the qPCR results, the RNA expression level of the target gene in each sample was calculated based on the Ct value, using the ΔΔCt relative quantification method. The relative expression level of the target gene is represented as 2 ^ΔΔCT , and all results are reported to five decimal places.

The calculation formulas are as follows:

ΔCT = Average Ct value of PCSK9 gene – Average Ct value of GAPDH gene

ΔΔCT = ΔCT (drug-treated group) – ΔCT (control group)

Relative mRNA expression level of target gene = 2^{–ΔΔCT}

Expression level relative to the control group = Relative mRNA expression level of PCSK9 to GAPDH for each sample / Relative mRNA expression level of PCSK9 to GAPDH in the control group

Inhibition rate (%) = (1 - expression level relative to the control group) × 100%.

Experimental Results

After processing the data according to the method described in section 4.7, the PCSK9 mRNA expression level of each biological replicate was normalized to the control group (expression level relative to the control group = PCSK9 mRNA expression level relative to GAPDH for each sample / PCSK9 mRNA expression level relative to GAPDH for the control group). The inhibition rate (%) = (1 - expression level relative to the control group) × 100%. The mean and standard deviation of the IC ₅₀ from three experiments were calculated and the means were ranked. The results are as follows.

Table 11 Analysis of qPCR Results for PCSK9 Inhibition IC ₅₀ in Primary Rat Hepatocytes

Figure 2 Dose-response curves of 13 candidate compounds in primary rat hepatocytes. The expression levels of the compounds at different concentrations relative to the control group were plotted, and the dose-response curves were fitted using the four-parameter logistic fitting function in GraphPad Prism software to calculate the IC ₅₀ . The horizontal axis represents drug concentration in nM; the vertical axis represents the inhibition rate of PCSK9 mRNA relative to the control group.

Table 12 IC ₅₀ ranking of qPCR results

After processing the data according to Section 4.7, the PCSK9 protein expression level of each biological replicate was normalized to the control group. The inhibition rate (%) = (mean expression level of the control group - mean expression level of the sample) / mean expression level of the control group × 100%. The average value and standard deviation of IC ₅₀ from three experiments were calculated and the means were ranked. The results are as follows.

Table 13 Analysis of ELISA Results for IC ₅₀ in Primary Rat Hepatocytes

Figure 3 Dose-response curves of 13 candidate compounds in primary rat hepatocytes. The expression levels of the compounds at different concentrations relative to the control group were plotted, and the dose-response curves were fitted using the four-parameter logistic function in GraphPad Prism software to calculate the IC ₅₀ . The horizontal axis represents drug concentration in nM; the vertical axis represents the inhibition rate of PCSK9 protein content relative to the control group.

Table 14 IC ₅₀ ranking of ELISA test results

The rankings of the qPCR IC ₅₀ experiment and the ELISA IC ₅₀ experiment were summarized, and the results are shown in the table below.

Table 15 Summary Table of qPCR and ELISA IC ₅₀ Data

According to the data summary table, D82-DV29PG5, D82RAT1-DV29PG5, Inclisiran, sh1-DV29PG5, and PCS-A2-DV29PG5L could not be fitted for IC ₅₀ due to the low upper limit of the fitting curve. The top five sequences in the overall ranking are D82RAT6-DV29PG5, D82RAT5-DV29PG5, D82RAT3-DV29PG5/D82RAT4-DV29PG5, D82RAT2-DV29PG5, and D82RAT7-DV29PG5, in order.

Change History

Appendix

Data Source