iPSC GMP SERVICES: iPSC Generation, Expansion, and Banking

cGMP iPSC Expansion and Banking
	Number of Vials	Cell Count	Other Deliverables
Master Cell Bank	50-200 vials	1x10^6 cells/vial	QC and CoA
Working Cell Bank	50-200 vials	1x10^6 cells/vial	QC and CoA

*Stocking of Frozen Cells (Annual Storage): 24/7 monitoring

 

Customized iPSC Generation (Episomal Method)
	Number of Clones	Number of Vials	Cell Count	Other Deliverables
Seed Bank	3 Clones	10 vials	1x10^6 cells/vial	QC Characterization and CoA

GMP SERVICES: iPSC Gene Editing

With 13+ years of stem cell & genome editing experience, ASC offers high-quality gene editing services using the latest technology, CRISPR and TARGATT^TM. For all CRISPR, fee-for service gene editing projects, the customer must obtain a CRISPR license. With ASC’s TARGATT^TM technology, customers can avoid having to obtain additional licensing and only need a single license from ASC!

iPSC Gene Editing
Service	Details
CRISPR/Cas9 Gene Editing	Customer needs to obtain CRISPR license ASC performs fee-for-service gene editing
TARGATT™ Gene Editing	License terms already in place
GMP-like Plasmid Production For Gene Editing	QC: purity, sequence, residual host DNA/protein/RNA test, etc.

In-Process QC:

• Reagent QC (plasmid map/sequence)
• Validation (cell, gRNA, integrase)
• Transfection pool genotyping
• Clone genotyping

TARGATT^TM Site-Specific Knockin Technology

Unlike other gene editing companies, ASC has a unique gene-editing technology, TARGATT^TM, that enables site-specific, irreversible gene insertion at a safe-harbor locus, H11 or ASC2. The unidirectional gene insertion at an active, intergenic locus does not disrupt other internal genes. TARGATT^TM works well in non-dividing cells and permits single-copy insertion of large fragments up to 20kb. ASC has observed high integration efficiencies and medium to high protein expression in stable cells.

The NIH awarded Applied StemCell with a $2M SBIR grant for the improvement of its hTARGATT^TM technology for human gene and cell therapy. According to the NIH ASC’s proprietary gene editing technology

 

 “could efficiently and safely treat more than ten thousand monogenic human diseases.”

                                                        - NIH grant review group summary statement

GMP SERVICES: iPSC DIFFERENTIATION

Integration-free protocols to differentiate iPSCs to various committed somatic lineages, including NK cells, T cells, RPE cells, neurons, and many others.

 

iPSC Differentiation
Service	Details
iPSC Differentiation: Customer provides protocol	Tech transfer Engineering run/scale up cGMP production
iPSC Differentiation: ASC’s SOP	Engineering run/scale up cGMP production

GMP SERVICES: iPSC DIFFERENTIATION TO COMMITED SOMATIC LINEAGES

 

GMP SERVICES: iPSC DIFFERENTIATION TO NK CELLS (iNK)

Characterization of the ASE-9708 NK Cells

Figure 1. Flow cytometry analysis of ASE-9708 iPSC-derived NK cells for NK cell biomarkers. Cryopreserved NKs, differentiated from Applied StemCell’s control iPSC line, ASE-9211 were recovered in NK culture media. The cells were stained with NK cell markers, CD45 and CD56 at day 2. Left: Isotype control antibodies. Right: CD45/CD56.

GMP SERVICES: iPSC DIFFERENTIATION TO RPE CELLS

Characterization of the ASE-9710 RPE Cells

Figure 2. Immunostaining of ASE-9710 iPSC-derived Retinal Pigment Epithelium Cells for retinal pigment
epithelium biomarkers. Cryopreserved RPE cells, differentiated from Applied StemCell’s control iPSC line, ASE9211 were recovered in RPE culture media. The cells were stained with RPE markers, PMEL1, MITF, ZO-1 and
RPE65.

TARGATT^TM Master iPSC Platform for Allogeneic Products

The comprehensive TARGATT^TM Master iPSC platform permits the safe and efficient insertion of any gene of interest at a safe harbor locus in the TARGATT^TM Master iPSCs. This process is facilitated by TARGATT^TM integrase, and the engineered cells can be further differentiated to various cells types.  

 

1. TARGATT^TM Master iPSCs

Pluripotent stem cells with a pre-engineered landing pad in a safe genomic locus for repeated, fast, and efficient gene knock-in by TARGATT^TM integrase

• Engineered from ASC’s GMP grade iPSC using TARGATT™ technology only (no CRISPR)
• All reagents are CGT grade (animal, serum-free)
• Follow GMP SOPs
• Documentation and QC/QA follow GMP SOP
• Certificate of Analysis provided
• All testing follows GMP requirement

2. iPSC Differentiation

For the creation of unlimited sources of off-the-shelf allogenic therapeutic cells.

• CAR-T or CAR-NK cells (no need for lentiviral transfection), neurons, or other cell types

TARGATT™ iPSC-iNK Platform in CAR-iNK Therapy

Current Unmet Needs Using CAR-T/NK Therapy

1. Autologous CAT-NK/T products can only be used by a single patient and it is expensive
2. Random CAR gene insertion caused by lentivirus or transposon methods is unsafe
3. CRISPR limits the payload size and has a low efficiency
4. Current lentivirus manufacturing of CAR-NK/T products

TARGATT™ iPSC-iNK in CAR-NK Therapy

Our TARGATT^TM system can be used to knock-in your CAR gene. ASC’s experts can confirm your iPS cells express CAR and differentiate your CAR-positive cells into NK cells or T cells.

 

CAR Knock-In

• CAR was inserted at >40% efficiency
• 10x better than CRISPR/Cas
• CAR-iPSC express CAR
• Transfection done by lipofectamine,
  eliminating viral manufacturing

 

CAR positive iPSC line express CAR

Figure 3: Flow cytometry analysis of iPSC lines screened for surface expression of CAR. Monoclonal antibodies labeled with a fluorescent molecule were used for screening. During the screening process, an iPSC line that expressed CAR was identified. (Images: Left: CAR negative iPSCs; Right: CAR positive iPSCs)

CAR positive iPSC-iNK cells express CAR

Figure 4: qRT-PCR data for expression levels at each stage of the iPSC to NK cell differentiation process.

Figure 5: qRT-PCR data for OCT4 and CD56 levels throughout the differentiation process, including the final CD56 stage.