AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

Methylation-Sensitive Restriction Enzyme Quantitative Polymerase Chain Reaction Enables Rapid, Accurate, and Precise Detection of Methylation Status of the Regulatory T Cell (Treg)-Specific Demethylation Region in Primary Human Tregs.

Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.

Regulatory T cell expressed MyD88 is critical for prolongation of allograft survival.

MyD88 signaling directly promotes T-cell survival and is required for optimal T-cell responses to pathogens. To examine the role of T-cell-intrinsic MyD88 signals in transplantation, we studied mice with targeted T-cell-specific MyD88 deletion. Contrary to expectations, we found that these mice were relatively resistant to prolongation of graft survival with anti-CD154 plus rapamycin in a class II-mismatched system. To specifically examine the role of MyD88 in Tregs, we created a Treg-specific MyD88-deficient mouse. Transplant studies in these animals replicated the findings observed with a global T-cell MyD88 knockout. Surprisingly, given the role of MyD88 in conventional T-cell survival, we found no defect in the survival of MyD88-deficient Tregs in vitro or in the transplant recipients and also observed intact cell homing and expression of Treg effector molecules. MyD88-deficient Tregs also fail to protect allogeneic bone marrow transplant recipients from chronic graft-versus-host disease, confirming the observations of defective regulation seen in a solid organ transplant system. Together, our data define MyD88 as having a divergent requirement for cell survival in non-Tregs and Tregs, and a yet-to-be defined survival-independent requirement for Treg function during the response to alloantigen.

Requirement for CD28 in Effector Regulatory T Cell Differentiation, CCR6 Induction, and Skin Homing.

The skin, similar to most nonlymphoid tissues, contains substantial numbers of T cells. Among these, memory T cells serve a sentinel role to protect against pathogens, and regulatory T cells (Tregs) terminate immune responses as a check against unrestrained inflammation. Previously, we created conditional knockout mice with Treg-specific deletion of CD28. Although these mice have normal numbers of Tregs, these cells have lower levels of CTLA-4, PD-1, and CCR6, and the animals develop systemic autoimmunity characterized by prominent skin inflammation. In this study, we have performed a detailed analysis of the skin disease in these mice. Our data show that Treg-expressed CD28 is required for optimal maturation of CD44(lo)CD62L(hi) central Tregs into CD44(hi)CD62L(lo) effector Tregs (eTregs), as well as induction of CCR6 among the cells that do become eTregs. Although CD28-deficient Tregs are able to regulate inflammation normally when injected directly into the skin, they fail to home properly to inflamed skin. Collectively, these results suggest a key role for CD28 costimulation in promoting a central Treg to eTreg transition with appropriate upregulation of chemokine receptors such as CCR6 that are required for tissue homing.

Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability.

Foxp3(+) regulatory T cells (Treg cells) are required for immunological homeostasis. One notable distinction between conventional T cells (Tconv cells) and Treg cells is differences in the activity of phosphatidylinositol-3-OH kinase (PI(3)K); only Tconv cells downregulate PTEN, the main negative regulator of PI(3)K, upon activation. Here we found that control of PI(3)K in Treg cells was essential for lineage homeostasis and stability. Mice lacking Pten in Treg cells developed an autoimmune-lymphoproliferative disease characterized by excessive T helper type 1 (TH1) responses and B cell activation. Diminished control of PI(3)K activity in Treg cells led to reduced expression of the interleukin-2 (IL-2) receptor α subunit CD25, accumulation of Foxp3(+)CD25(-) cells and, ultimately, loss of expression of the transcription factor Foxp3 in these cells. Collectively, our data demonstrate that control of PI(3)K signaling by PTEN in Treg cells is critical for maintaining their homeostasis, function and stability.

MyD88 is essential to sustain mTOR activation necessary to promote T helper 17 cell proliferation by linking IL-1 and IL-23 signaling.

Myeloid differentiation primary response protein 88 (MyD88) is classically known as an adaptor, linking TLR and IL-1R to downstream signaling pathways in the innate immune system. In addition to its role in innate immune cells, MyD88 has been shown to play an important role in T cells. How MyD88 regulates helper T-cell differentiation remains largely unknown, however. Here we demonstrate that MyD88 is an important regulator of IL-17-producing CD4(+) T helper cells (Th17) cell proliferation. MyD88-deficient CD4(+) T cells showed a defect in Th17 cell differentiation, but not in Th1 cell or Th2 cell differentiation. The impaired IL-17 production from MyD88-deficient CD4(+) T cells is not a result of defective RAR-related orphan receptor γt (RORγt) expression. Instead, MyD88 is essential for sustaining the mammalian target of rapamycin (mTOR) activation necessary to promote Th17 cell proliferation by linking IL-1 and IL-23 signaling. MyD88-deficient CD4(+) T cells showed impaired mTOR activation and, consequently, reduced Th17 cell proliferation. Importantly, the absence of MyD88 in T cells ameliorated disease in the experimental autoimmune encephalomyelitis model. Taken together, our results demonstrate that MyD88 has a dual function in Th17 cells by delivering IL-1 signaling during the early differentiation stage and integrating IL-23 signaling to the mTOR complex to expand committed Th17 cells.