Haplobanking and the iPS Cell Stock Project​

​

▣ Haplobanking is still considered a more feasible immunogenicity strategy for domestic therapeautic coverage in Japan than genome-editing. Donor screening is more laborious, and the projected demographic coverage is less (40% vs. >90%), but HLA-homozygous line risk uncertainties are lower relative to genome-edited lines.

​

▣ Haplobanked lines were created using episomal plasmid reprogramming performed with plasmids donated by the Yamanaka lab to Addgene in 2012. The cocktail consisted of Oct3/4, Sox2, Klf4, MycL1, Lin28, EBNA1, and the dominant-negative form of TP53. Although episomal plasmid reprogramming is intended to be integration-free, residual plasmids were found in some cases. A single cell/well plating and limiting dilution process is believed to provide sufficient screening against residual vectors. Sendai viruses may offer reduced residuality risk, but their advantages remain to be tested.

​

▣ Chromosome 11 uniparental disomy (UPD) was detected in some reprogrammed lines. This may be attributable to p53 inhibition; nonetheless, p53 inhibition is desirable as a reprogramming efficiency enhancer. The cause remains unknown, but it was observed that the appearance of chromosome 11 UPD depended on the quality control (QC) method used; namely, it affected lines from donors RJWI, YZWJ, ILCL, and GLK (Method 3), but not from QHJI (Method 1).

​

▣ CiRA Foundation performs trilineage differentiation assays, and now, in response to user demands, also performs a cardiac differentiation potential assay. CiRA Foundation is willing to consider performance of further differentiation assays.

​

▣ During QC, cells were screened out for undifferentiated marker (e.g., TRA-1-60) underexpression. The cause of TRA-1-60 underexpression remains unknown, but it is posited that it is an artifact of sample timing.

​​

HLA Cloaking and Genome-Edited iPSC Lines​

​

▣ CiRA Foundation employs an HLA-C-retained knockout strategy for genome-edited lines. It is believed that this approach is advantageous relative to β2-microglobulin knockout, as the latter poses risks of precipitating 'missing-self recognition' NK cell responses.

​

▣ Performance of xenograft immunogenicity tests for genome-edited cells (e.g., in humanized murine models or non-human primate models) remains limited by lack of full recapitulation of the human immune system. In the case of the testing of HLA-C-retained lines, tests for T, B, and NK cells were performed separately with supplemental cytokines for each. HLA-C-*07:02 was tested in this manner and performed favorably. It is expected that a HLA-C-*07:02-retained iPSC line, in addition to 11 more HLA-C-retained lines, can provide >90% coverage for a collection of international demographic groups. However, tests for these remaining 11 lines remain to be done (though some unpublished data is reported to exist). 

​

▣ At present, it is expected that design of custom sgRNAs may still be required for genome-edited lines, given the variability of the exons on which consensus regions are located.

​

▣ Genome-edited lines have been subject to genomic integrity analysis and have not been found to exhibit off-target effects from CRISPR-Cas9 knockouts. Detected single nucleotide variants (SNVs) or insertion-deletions (indels) are attributed to mutagenicity arising during the cell culturing process. Minimization of passage number and culture time is sought to attenuate mutagenicity risk.

​

▣ Overall, the hypoimmunity advantage of HLA class II knockout remains an open question. HLA class II knockout may be needed for certain anti-cancer therapies involving allografts of antigen-presenting cells. It is expected that minor antigens can be ignored.

​

▣ Genome-edited lines are currently viewed as a strategy complementary to haplobanking, with the potential to provide coverage to patients for whom HLA-homozygous donors are not available.

​

Further Trends

​​

▣ It is still suspected that immune-privileged tissues, such as those protected by the blood-brain-barrier or blood-retina barrier, are amenable to iPS cell therapies without implementation of significant hypoimmunity strategies. (SciSci also notes that if one looks at the current CiRA-affiliated iPSC therapy clinical trials, they typically pertain to therapies concerning potentially immune-privileged tissues and organs. Examples include macular degeneration, Alzheimer's, and articular cartilage damage.)

​

▣ International regulatory consistency on genome-edited iPS cells is sought. So far as regulatory dialogue is concerned, the International Society for Stem Cell Research (ISSCR) serves as an intermediary forum between academia, government, and research. 

​

​​​

​

​