Genetic engineering and quality control ofclinical grade iPS cells as a source of HLCN061
1)
1)〇Kodai Saitoh , Yuma Fukutani ,1)Norihiro Tsuneyoshi ,1)
1)
Mitsuhiro Iwasaki , Ayaka Suga ,1)
Tomonori Hosoya ,Hironobu Kimura1) and Kouichi Tamura1)
1) Kobe Research Institute, HEALIOS K.K.
Abstract

Methods
Fig. 1 : Strategy of Generating Engineered iPSC expressing five factors

HLCN061 is a human iPSC derived NK cells differentiated from

Parental iPSC
(clinical-grade)clinical grade iPSC, in which IL15, CCR2B, CCL19, CD16a, andNKG2D have been introduced using PiggyBac system. After singlecell cloning, an iPSC clone expressing all 5 factors was selected forfurther application. Frozen Seed-Stock cells of the iPSC clone

Engineered iPSC
(clinical-grade)

Engineered iPSC
(clinical-grade)

Engineered iPSC
(clinical-grade)

Polyclonal pool

Monoclonal cell line

Seed-Stock

IL-15

showed negative in mycoplasma, bacterial and endotoxin tests.
G-banding karyological test with the iPSC Seed-Stock showed

CD16a (F176V)
NKG2D

normal human karyotype. RAISING method, PCR amplification
followed by high-throughput sequencing, unveiled that the PiggyBacScreening

Clone
A・qRT-PCR
・Flow cytometry
・ELISA

Clone
B

CCR2B
CCL19

SCC

donor DNAs have been integrated in the genome at total 13 locations.
No deleterious mutation that could increase tumorigenicity risk wasValidation

Clone
C

Stock

Clone
D

found in the iPSC Seed-Stock compared to parental iPSC through
WGS analysis followed by SNV/INDEL/CNV calling coupled with・・・・・・・Karyotype
・Insertion sites
・Copy number・WGS(SNV/INDEL/CNV)・RNA-seqetcRNA-seq expression data. We have concluded that this iPSC SeedStock is suitable as a source material of iPSC MCB, followed byClinical grade parental iPSCs were transfected with expression vectors that contained each cDNA

iPSC WCB and HLCN061. Additional analyses will be conducted inthese iPSC banks as well as HLCN061 to assess tumorigenicity riskcloning, we selected an iPSC clone expressing all 5 factors by qRT-PCR, flow cytometry and ELISA.

(IL15, CCR2B, CCL19, CD16a (F176V), and NKG2D) using PiggyBac method. After single-cellof HLCN061.

PiggyBac method was used tointegrate transgenes

We further performed validation tests for frozen Seed-Stock cells of the iPSC clone.
SCC = Single Cell Cloning; WGS = Whole Genome Sequence; SNV = Single Nucleotide Variation;
INDEL = Insertion/Deletion; CNV = Copy Number Variation

Results(1) Selection of candidate as a source of HLCN061(3) Quality Control of the iPSC Seed-Stock (Clone G)

Fig. 2 : Expression of each transgene in selected iPSC clones

Table 2 : Summary of validation tests
Test
Mycoplasma
Bacteriology
Endotoxin
KaryotypeSpecificationNegative
Negative≤ 5.00 EU/mL
46 XY, Normal

Residual vector test

≤ 1 copy/100 cells

Undifferentiation
stem cell marker
CD16

NKG2D

CCR2B

IL-15

CCL19Results
Negative
Negative< 0.50 EU/mL
46 XY, Normal≤ 1 copy/100 cells(Undetectable)
SSEA-4 > 99%
TRA-1-60 > 99%
TRA-1-81 > 99%
OCT-4A > 99%
Done (as described in (2))
Done (as described in (2))
Done (as follows)

SSEA-4 >70%
TRA-1-60>70%
TRA-1-81 >70%
OCT-4A >70%
For information
For information
For information

H, Clone I, and Clone J). The level of IL-15 and CCL19 in the culture supernatant was measured by ELISA.

Insertion sites
Copy number
Tumorigenicity

Clone G secretes more IL-15 than the other four clones (Clone E, Clone H, Clone I, and Clone J).

The safety and quality tests for clinical-grade engineered iPSC Seed-Stock are listed in the table above:

Expression level of transgenes in the clonal lines as measured by flow cytometry and ELISA. Transgenes(CD16a, NKG2D, and CCR2B) are highly expressed on cell surface of five clones (Clone E, Clone G, Clone(2) Identification of integration sites and copy number
Table 1: Integration sites and copy number of transgenes in the Clone G
Chromosome : Position
Chr1 : XXX,XXX,XXX
Chr1 : XXX,XXX,XXX

Direction
forwardreverseTransgene
XXX
XXX

Chr1 : XXX,XXX,XXX

forwardXXX

Chr7 : XXX,XXX,XXX

forwardXXX

Chr9 : XXX,XXX,XXX
Chr11 : XXX,XXX,XXX
Chr12 : XXX,XXX,XXX
Chr14 : XXX,XXX,XXX
Chr18 : XXX,XXX,XXX
Chr18 : XXX,XXX,XXX

reversereversereversereverseforwardreverseXXXXXX

WGS analysis (CNV)

No CNV was foundin the iPSC Seed-Stock!!

WGS analysis (SNV/INDEL)

RNA-seq
TPM(Seed-Stock)

TPM(Parent)

Gene X

3.55

2.78

Gene Y intron

Gene Y00

Gene Z intron

Gene Z

2.62

3.89

Chromosome : Position Annotation

Gene

Chr1 : XXX,XXX,XXX

Gene X intron

Chr7 : XXX,XXX,XXX
Chr17: XXX,XXX,XXX

We analyzed aligned sequences obtained from the iPSC Seed-Stock (Clone G) compared to parental iPSC
through WGS. CNV analysis highlighted 0 variant. SNV/INDEL analysis highlighted 0 variant for impact

XXX

score HIGH and 3 SNV/INDEL for MODERATE by using SnpEff (version 5.1) in cancer-related genes

XXX

intron did not result in more than 2-fold expression change of mRNA analyzed by RNA-seq. These results

XXX

forwardXXX

Chr20 : XXX,XXX,XXX

forward

XXX

reverseRAISING method

Table 3 : Risk assessment scheme by NGS data analysis

XXX

Chr18 : XXX,XXX,XXX

ChrX : XXX,XXX,XXX

NumberTransgeneof sites
IL15
2CCL19
3
CCR23
CD161
NKG2D
2DAP102(4) Tumorigenicity risk assessment of the iPSC Seed-Stock (Clone G)

XXX

In the Clone G, total 13 integration sites were identified by RAISING method (Wada Y, et, al. 2022, Commun Biol).
12 integration sites except for chr X were heterozygous insertion confirmed by WGS. The copy number of eachtransgene was analyzed using the Droplet Digital PCR (ddPCR) method. Genomic DNA PCR followed by Sanger
Sequencing identified which cDNA is inserted at which integration sites (data undisclosed).

(COSMIC Tier1 v97 or Shibata’s List). All the SNV/INDEL are located at intron region. The SNVs found inindicate that No deleterious mutation that could increase tumorigenicity risk was found in the iPSC Seed-Stockcompared to parental iPSC.

Conclusion
We have concluded that the iPSC Seed-Stock (Clone G) is suitable as a source
material of iPSC MCB, followed by iPSC WCB and HLCN061. Additional
analyses will be conducted in these iPSC banks as well as HLCN061 to assess
tumorigenicity risk of HLCN061.
演題名：Genetic engineering and quality control of clinical grade iPS cells as a source of HLCN061
施設名：株式会社ヘリオス神戸研究所 氏 名：齋藤浩大

筆頭演者は株式会社ヘリオスの社員です。