TARGATT™ RUO Hypo Plus Human iPSC

A characterized iPSC starting point for allogeneic and multilineage immune-evasion research.

• Favorable development profile—reprogrammed from CD34+ cord blood cells for reduced immunogenicity and fewer somatic mutations compared to reprogrammed adult cells
• Hypoimmunogenic—B2M/CIITA double knock-out eliminates expression of HLA Class I and II molecules to avoid recognition by CD8+ and CD4+ T cells
• Five complementary immune-modulatory knock-ins—HLA-F-B2M, CD200, CCL21, HLA-G-B2M and SERPINB9 knock-ins available
• Large knock-in ready—reliable H11 safe harbor locus using TARGATT large knock-in technology

Overview

One common strategy for engineering hypoimmune cells is to disrupt genes involved in HLA expression using B2M/CIITA double knock-out, which eliminates the expression of HLA Class I and II molecules.

But HLA knockout alone might not be enough. Natural killer (NK) cells provide another layer of immune surveillance. Unlike conventional T cells, NK cells can become activated when they encounter cells with abnormally low levels of HLA Class I. And this creates an important engineering challenge: reducing HLA Class I expression may decrease T-cell recognition, but the same modification can potentially increase susceptibility to NK-cell-mediated killing.

Rather than relying exclusively on HLA reduction, AST-9750 TARGATT™ Hypo Plus Human iPSC combines two targeted knockouts—B2M and CIITA—with five complementary immune-modulatory knock-ins that address complementary immune pathways.

This product also harnesses the power of the TARGATT™ platform, which means you can insert all these genes in a single reaction—up to 20 kb—with additional DNA over 20 kb added in nested reactions. And its placement at the H11 safe harbor locus avoids the potential for silencing and harmful random integration that you get with lentivirus and transposon-mediated integration and consistently delivers gene expression*.

How it works

AST-9750 TARGATT™ Hypo Plus Human iPSC is a starting platform designed to address multiple mechanisms involved in allogeneic immune recognition and clearance by combining two targeted knockouts with five complementary immune-modulatory knock-ins.

B2M and CIITA double knockout

The cell surface expression of HLA I and HLA II molecules is eliminated by knocking out two genes (Figure 1):

• B2M, which encodes the ß2-microglobulin protein, a key part of the HLA class I complex
• CIITA, the HLA II transactivator which is essential for transcription of HLA class II genes

Figure 1. Knocking out cell surface expression of HLA I and HLA II eliminates the T cell response, although other gene edits are needed to avoid innate immune responses.

Non-Classical HLA Signaling

HLA-F-B2M and HLA-G-B2M are two of the complementary immune-modulatory knock-ins incorporated to address innate immune responses associated with reduced conventional HLA expression, including NK-cell activity.

Myeloid and Immune Modulation

The CD200 knock-in provides an additional immunoregulatory mechanism relevant to interactions with myeloid immune cells, while the CCL21 knock-in adds another dimension intended to influence immune-cell recruitment and the local immune environment.

Cytotoxic Resistance

SERPINB9 is an endogenous inhibitor of granzyme B, a cytotoxic protease used by cytotoxic T lymphocytes and NK cells. Its inclusion as a complementary immune-modulatory knock-in provides an additional design element intended to address granzyme B-mediated cytotoxicity.

Together, these targeted knockouts and complementary immune-modulatory knock-ins are designed to address multiple adaptive and innate immune pathways.

Learn more about our approach to developing hypoimmunogenic hiPSCs by watching our webinar, “The Future of Allogeneic Therapy: ASC’s Hypoimmunogenic Donor Cells.”

Figure 2. TARGATT™ Hypo Plus Human iPSC combines targeted knockouts and immune-modulatory knock-ins to address multiple immune pathways.