Neuronal TDP-43 aggregates are a hallmark ALS pathology. The integrated stress response (ISR) occurs downstream of TDP-43 pathology and may promote neurodegeneration. Here we demonstrate that a CNS penetrant small molecule eIF2B activator inhibits the ISR in cellular models of ALS and the brain of an inducible mouse model of TDP-43 pathology, where it transiently slowed progression of locomotor deficits and neurodegeneration. ISR activation was observed in ALS patient spinal cord and CSF. The investigational drug DNL343 was advanced into Phase 1 and Phase 1b randomized, double-blind, placebo-controlled trials in healthy and ALS participants, respectively (NCT04268784/NCT05006352); the primary objective in both studies was to investigate the safety and tolerability DNL343. DNL343 demonstrated a half-life supporting once-daily dosing and showed extensive CSF distribution. DNL343 was generally well tolerated and reduced ISR biomarkers in peripheral blood mononuclear cells and CSF of ALS participants. Therefore, DNL343 is a useful investigational drug to explore the effects of ISR inhibition in ALS models and individuals with neurological diseases.

The integrated stress response (ISR) is a conserved pathway in eukaryotic cells that is activated in response to multiple sources of cellular stress. Although acute activation of this pathway restores cellular homeostasis, intense or prolonged ISR activation perturbs cell function and may contribute to neurodegeneration. DNL343 is an investigational CNS-penetrant small molecule ISR inhibitor designed to activate the eukaryotic initiation factor 2B (eIF2B) and suppress aberrant ISR activation. DNL343 reduced CNS ISR activity and neurodegeneration in a dose-dependent manner in two established in vivo models – the optic nerve crush injury and an eIF2B loss of function (LOF) mutant – demonstrating neuroprotection in both and preventing motor dysfunction in the LOF mutant mouse. Treatment with DNL343 at a late stage of disease in the LOF model reversed elevation in plasma biomarkers of neuroinflammation and neurodegeneration and prevented premature mortality. Several proteins and metabolites that are dysregulated in the LOF mouse brains were normalized by DNL343 treatment, and this response is detectable in human biofluids. Several of these biomarkers show differential levels in CSF and plasma from patients with vanishing white matter disease (VWMD), a neurodegenerative disease that is driven by eIF2B LOF and chronic ISR activation, supporting their potential translational relevance. This study demonstrates that DNL343 is a brain penetrant ISR inhibitor capable of attenuating neurodegeneration in mouse models and identifies several biomarker candidates that may be used to assess treatment responses in the clinic.

Genetic engineering of allogeneic cell therapeutics that fully prevents rejection by a recipient’s immune system would abolish the requirement for immunosuppressive drugs or encapsulation and support large-scale manufacturing of off-the-shelf cell products. Previously, we generated mouse and human hypoimmune pluripotent (HIP) stem cells by depleting HLA class I and II molecules and overexpressing CD47 ( B2M −/− CIITA −/− CD47 + ). To determine whether this strategy is successful in non-human primates, we engineered rhesus macaque HIP cells and transplanted them intramuscularly into four allogeneic rhesus macaques. The HIP cells survived unrestricted for 16 weeks in fully immunocompetent allogeneic recipients and differentiated into several lineages, whereas allogeneic wild-type cells were vigorously rejected. We also differentiated human HIP cells into endocrinologically active pancreatic islet cells and showed that they survived in immunocompetent, allogeneic diabetic humanized mice for 4 weeks and ameliorated diabetes. HIP-edited primary rhesus macaque islets survived for 40 weeks in an allogeneic rhesus macaque recipient without immunosuppression, whereas unedited islets were quickly rejected.

Eukaryotic translation initiation factor 2B (eIF2B) is a key component of the integrated stress response (ISR), which regulates protein synthesis and stress granule formation in response to cellular insult. Modulation of the ISR has been proposed as a therapeutic strategy for treatment of neurodegenerative diseases such as vanishing white matter (VWM) disease and amyotrophic lateral sclerosis (ALS) based on its ability to improve cellular homeostasis and prevent neuronal degeneration. Herein, we report the small-molecule discovery campaign that identified potent, selective, and CNS-penetrant eIF2B activators using both structure- and ligand-based drug design. These discovery efforts culminated in the identification of DNL343, which demonstrated a desirable preclinical drug profile, including a long half-life and high oral bioavailability across preclinical species. DNL343 was progressed into clinical studies and is currently undergoing evaluation in late-stage clinical trials for ALS.

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

Transplantation of allogeneic pancreatic donor islets has successfully been performed in selected patients with difficult-to-control insulin-dependent diabetes and impaired awareness of hypoglycemia (IAH). However, the required systemic immunosuppression associated with this procedure prevents this cell replacement therapy from more widespread adoption in larger patient populations. We report the editing of primary human islet cells to the hypoimmune HLA class I- and class II-negative and CD47-overexpressing phenotype and their reaggregation into human HIP pseudoislets (p-islets). Human HIP p-islets were shown to survive, engraft, and ameliorate diabetes in immunocompetent, allogeneic, diabetic humanized mice. HIP p-islet cells were further shown to avoid autoimmune killing in autologous, diabetic humanized autoimmune mice. The survival and endocrine function of HIP p-islet cells were not impaired by contamination of unedited or partially edited cells within the p-islets. HIP p-islet cells were eliminated quickly and reliably in this model using a CD47-targeting antibody, thus providing a safety strategy in case HIP cells exert toxicity in a future clinical setting. Transplantation of human HIP p-islets for which no immunosuppression is required has the potential to lead to wider adoption of this therapy and help more diabetes patients with IAH and history of severe hypoglycemic events to achieve insulin independence.

Off-the-shelf CAR T cells potentially offer advantages over autologous strategies such as ease of manufacturing, quality control, off-the-shelf availability, and lack of T cell dysfunction, as well as the ability to generate a more consistent CAR T product from healthy T cells. However, the vigorous host-versus-graft immune response against histoincompatible T cells prevents expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced with this approach. CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), and we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are disrupted and CD47 is over-expressed. We describe here the engineering of human immune evasive CAR T cells building on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118).Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas12b technology was used to disrupt the B2M, CIITA, and TCR genes, and lentiviral transduction was used to overexpress CD47 and to express a CD19 CAR to generate hypoimmune (HIP) CD19 CAR T cells. Control T cells were unmanipulated except for overexpression of the CD19 CAR (unmodified).For 3 months persistence studies, allogeneic SGM3 humanized mice were injected with 1 × 106 Luc+ Nalm6 cells and received 7 × 106 control CD19 CAR T cells or HIP CD19 CAR T cells.In the mice treated with either unmodified CD19 CAR T cells and HIP CD19 CAR T cells, tumor control was initially rapidly achieved. However, unmodified CD19 CAR T cells were eventually rejected by the host and the loss of these cells resulted in re-growth of tumor. By contrast, in HIP CD19 CAR T injected mice, tumor control was maintained throughout the study, including following a rechallenge at day 83 with NALM6 cells without further administration of HIP CD19 CAR T cell. Flow cytometry at endpoint from bone marrow and spleen confirmed persistence of HIP CD19 CAR T cells.These findings show that HIP CD19 CAR T cells are immune evasive in allogeneic recipients and data suggest that HIP CD19 CAR T cells are able to persist and maintain efficacy without immunosuppression. Citation Format: Xiaomeng Hu, Pascal Beauchesne, Karl Manner, Corie Gattis, Priscilla Ngo, Rowena De Jesus, Ramya Ankala, Chi Young, Frank Wells, Lindong Weng, Kathy White, William E. Dowdle, Aaron Foster, Terry J. Fry, Sonja Schrepfer. Engineered hypoimmune CAR T cells provide lasting tumor control in immunocompetent allogeneic humanized mice even with re-challenge. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4091.

Improved differentiation protocols allow large-scale generation of induced pluripotent stem cell (iPSC) -derived islets. In order to make allogeneic “off-the-shelf” islets clinically useful and avoid rejection absent immunosuppression (IS) , they need to evade host immune responses, a property we refer to as being “hypoimmune.” Rhesus macaque (NHP) iPSCs were engineered to knock-out function of MHC class I and II and overexpress CD47 (HIP iPSCs) . HIP iPSCs and unedited iPSCs (wt iPSCs) were transduced to express luciferase and transplanted IM into four allogeneic NHPs per group. There was a strong IFN-γ ELISpot T cell response 1 week after wt iPSC transplantation, and increased production of wt-iPSC specifc IgM antibodies (Ab) and IgG Abs. At all time points tested (up to 12 weeks) , PBMCs killed wt iPSCs via direct and Ab-mediated cellular cytotoxicity. Furthermore, serum from NHPs killed wt iPSCs via complement-dependent cytotoxicity. In contrast, NHPs that received HIP iPSCs showed no measurable immune response against HIP iPSCs at all time points. After 6 weeks, NHPs initially receiving wt iPSCs were injected with HIP iPSCs. Although the NHPs maintained their strong immune response against wt iPSCs, they did not mount any response against HIP iPSCs. NHPs receiving HIP iPSCs first developed a strong cellular and Ab-response against the subsequently injected wt iPSCs but continued to have no reactivity against HIP iPSCs. Bioluminescence imaging in vivo revealed rejection of all wt iPSC grafts in both groups within 2-3 weeks after transplantation, while all HIP iPSC grafts survived the study period of 16 weeks. As a step to extending these studies to human iPSCs, we created human HIP iPSCs. We were able to differentiate these human HIP iPSCs into functional HIP islet cells that evaded host immune responses. This work suggests that HIP modifications do not impact islet cell differentiation or function. These data support the development of allogeneic HIP islets for the treatment of patients with T1DM. Disclosure X.Hu: None. F.Wells: None. T.Mcgill: Employee; Sana Biotechnology Inc. M.Rukstalis: Employee; Sana Biotechnology Inc. C.Gattis: None. R.Basco: None. J.R.Millman: Consultant; Sana Biotechnology Inc., Other Relationship; Sana Biotechnology Inc., Vertex Pharmaceuticals Incorporated, Research Support; Sana Biotechnology Inc., Stock/Shareholder; Salentra Biosciences. S.Schrepfer: Other Relationship; Sana Biotechnology Inc. K.White: None. A.G.Olroyd: None. R.Dejesus: None. A.Dominguez: Employee; Sana Biotechnology Inc. W.E.Dowdle: Employee; Sana Biotechnology Inc., Stock/Shareholder; Sana Biotechnology Inc. E.Y.L.Chu: None. A.Friera: None. C.Young: Employee; Sana Biotechnology Inc.

Off-the-shelf CAR T cells potentially offer advantages over autologous strategies such as ease of manufacturing, quality control, avoidance of malignant contamination and T cell dysfunction, as well as the ability to generate a more consistent CAR T product from healthy T cells. However, the vigorous host-versus-graft immune response against histoincompatible T cells prevents expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced with this approach. CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), and we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are disrupted and CD47 is over-expressed. We describe here the engineering of human immune evasive CAR T cells building on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas12b technology was used to disrupt the B2M, CIITA, and TCR genes, and lentiviral transduction was used to overexpress CD47 and to express a CD19 CAR to generate hypoimmune (HIP) CD19 CAR T cells. Control T cells were unmanipulated except for overexpression of the CD19 CAR. When transplanted into allogeneic humanized mice, hypoimmune CAR T cells evade immune recognition by T cells using ELISPOT analysis. In contrast, transplantation of control CD19 CAR T cells generated from the same human donor resulted in a significant T cell activation (p<0.0001 unpaired t-test). Innate immune cell assays show that CD47 overexpression protects HLA-I/II deficient CD19 CAR T cells from NK cell and macrophage killing in vitro and blocking of CD47 results in killing by innate allogeneic immune cells. For persistence studies, allogeneic humanized mice were injected with 1´106 Luc+ Nalm6 cells and received 7´106 control CD19 CAR T cells or HIP CD19 CAR T cells. However, CD19 CAR T cells are recognized and rejected by the allogeneic immune system of the humanized mice, leading to tumor grow in this group. Flow cytometry on Day 56 from bone marrow and spleen confirms persistence of HIP CD19 CAR T cells, but not control CD19 CAR T cells (see Figure). These findings show that HIP CD19 CAR T cells are functionally immune evasive in allogeneic recipients with cytotoxic anti-tumor capacity and suggest they could provide universal HIP CAR T cells that are able to persist without immunosuppression. Furthermore, these data suggest that HIP CD19 CAR T cells can be used for re-dosing strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction: Allogeneic CAR T cell therapies may offer key advantages over autologous therapies including choice of starting cells, better manufacturing control, and lower cost. However, the manufacture of these cells imposes a high demand on genome engineering specificity and efficiency, given the dual needs for product safety and yield. This challenge is amplified for newer generations of allogeneic CAR T cell designs that may offer improved performance but require more edits to produce, which may increase the potential of unintended genome modification and insufficient yield of fully engineered product. Here we have tested a novel editing platform, Cas12b, for the ability to mediate efficient and specific disruption of three genes (B2M, CIITA, and TRAC) during large scale manufacturing of CAR T cells with immune evasive properties and abrogated graft-vs-host response. We have characterized the resulting cell product for on-target modification efficiency, type and levels of unintended genome modification, and potency for tumor clearance in a Nalm6 humanized mouse model, with the aim of demonstrating suitability for translation to human studies. Methods: Our manufacturing strategy to generate hypoimmune CAR T cells introduces five genetic modifications, using Cas12b for disruption of B2M, CIITA, and TCR expression, and lentiviral transduction to overexpress CD47 and introduce a CD19 CAR. T cells obtained from healthy donors were first transduced with lentiviral vector and then edited simultaneously to disrupt the B2M, CIITA and TRAC genes via electroporation of Cas12b mRNA and appropriate guides. Edited and purified cells were characterized via flow cytometry for efficiency of each genetic modification. Cells were further characterized for knockout levels via next generation sequencing (NGS) as well as vector copy number per diploid genome (VCN). Cells were characterized for viability, proliferation, and efficacy in a Nalm6 mouse model of tumor clearance. An initial assessment of chromosomal translocations was performed by G banding karyotype analysis. To assess editing specificity, each Cas12b guide was evaluated by Cas-OFFinder and an unbiased oligonucleotide duplex capture assay in T cells from multiple donors under conditions of high on-target modification. Candidate off-target sites were then examined for evidence of cleavage via PCR amplification from the manufactured cell product followed by NGS. Results: Our manufacturing process yielded clinically relevant scales of cell product having > 85% B2M knockout, > 90% CIITA knockout, and < 1% of residual TCR expressing cells. VCN levels were < 5. Cell viability and proliferation were minimally impacted by the gene editing process. Cells exhibited robust anti-tumor properties with doses of 7 x 106 hypoimmune CD19 CAR T cells (per animal) providing efficient killing in a Nalm6 mouse model. Initial karyotype analysis identified low levels of translocation between on-target sites with overall translocation levels otherwise not significantly affected compared to unedited cells. Our characterization of Cas12b in edited T cells across multiple donors revealed high specificity, consistent with previous reports (Nat Commun 10 212), with studies to date having yielded no evidence of cleavage at any candidate off-target site. Summary: Cas12b-mediated multi-locus editing of allogeneic CAR T cells during large scale manufacture resulted in a high yield product with potent tumor clearance capabilities. Initial analysis of editing specificity across multiple donors and engineering runs identified no off-target sites, and early karyotype assessments indicated an overall level of chromosomal translocations consistent with prior experiences in the field. In companion in vivo studies, these hypoimmune CD19 CAR T cells have been shown to efficiently evade host immune response (see posters by Hu and Duback). Together, these data demonstrate a profile for potency, genome integrity, and manufacturing yield that supports progressing these cells into human studies.

Off-the-shelf CAR T cells may offer advantages over autologous strategies, including ease of manufacturing, improved quality control with avoidance of malignant contamination and T cell dysfunction, and the ability to generate a final product from healthy T cells. However, host-versus-graft immune response against histoincompatible T cells prevents the expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced. While T cells express CD47, we demonstrate here that CD47 expression above endogenous levels is important for immune evasion. We describe here the engineering of human immune evasive CAR T cells building on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). The goal is to achieve improved rates of durable complete remissions by improving allogeneic CD19CAR persistence, since it has been shown that autologous CAR T cells have greater durability over years than allogeneic CAR T cells. Human T cells from healthy donors were obtained by leukapheresis. To generate hypoimmune CD19CAR T cells, gene editing was used to eliminate HLA-I/II and TCR expression and lentiviral transduction was used to express CD47 and CD19CAR containing a 4-1BB costimulatory domain to generate hypoimmune CD19CAR T cells. Control CD19CAR T cells were unmanipulated, i.e., unedited, except for lentiviral transduction used to express CD19CAR. Hypoimmune CD19CAR T cells persist in allogeneic humanized mice and lack T cell activation measured using bioluminescence imaging and ELISPOT analysis, respectively. In contrast, transplantation of control CD19CAR T cells generated from the same human donor resulted in rejection (ELISPOT mean 59 and 558 spot frequencies for hypoimmune CD19CAR T cells and control CD19CAR T cells, respectively; p<0.0001 unpaired t-test). Innate immune cell assays show that CD47 overexpression protects hypoimmune CD19CAR T cells from NK cell and macrophage killing. A blocking antibody against CD47 made the hypoimmune CD19CAR T cells susceptible to macrophage and NK cell killing, confirming the importance of CD47 overexpression to evade innate immune clearance. Importantly, CD47 seemed to provide protection from all NK cell populations while other tested NK cell inhibitory molecules (such as HLA-E/G, PD-L1) seemed to prevent NK cell killing of only certain subpopulations rather than primary NK cells in total. Hypoimmune CD19CAR T cells retain their antitumor activity in the Nalm-6 B cell leukemia model in vitro and in vivo comparable to control CD19CAR T cells derived from various donors. Thus, hypoimmune edits seem to not impact CD19CAR T cell activity and have the potential to provide universal CAR T cells that are able to persist without immunosuppression. Citation Format: Xiaomeng Hu, Kathy White, Corie Gattis, Ryan Clarke, Sam Landry, Ron Basco, Eleonore Tham, Emily Luo, Andrew Tucker, Christopher Bandoro, Elaine Chu, Chi Young, Karl Manner, Priscilla Nho, Ben Lam, Pascal Beauchesne, Aaron Foster, William E. Dowdle, Edward J. Rebar, Terry J. Fry, Sonja Schrepfer. Engineered hypoimmune allogeneic CAR T cells as potential off-the-shelf CAR T cell immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5598.

Off-the-shelf CAR T cells could offer advantages over autologous strategies, including ease of manufacturing, quality control and avoidance of malignant contamination and T cell dysfunction. TCR editing can effectively prevent graft-versus-host reactions. However, the vigorous host-versus-graft immune response against histoincompatible T cells prevents expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced. CD47 overexpression can block both NK cell and macrophage killing (J Exp Med (2021) 218 (3): e20200839), and we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are inactivated and CD47 is over-expressed. We describe here the engineering of human immune evasive CAR T cells based on our previously described hypoimmune technology. Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas9 technology was used to delete b2m, CIITA, and TCR and lentiviral transduction to overexpress CD47 and CD19CAR. Control T cells were unmanipulated except for overexpression of CD19CAR containing a 41BB costimulatory domain. When transplanted into allogeneic humanized mice, hypoimmunogenic HLA-I/II- TCR- CD47+ CD19CAR+ T cells evade immune recognition by T and B cells compared to CD19CAR+ T cells generated from the same human donor using ELISPOT and flow cytometry analysis. Innate immune cell assays show that CD47 overexpression protects HLA-I/II deficient CAR T cells from NK cell and macrophage killing in vitro and in vivo. Relative CD47 expression levels were analyzed to understand the relevance of CD47 for protection from macrophage and NK cell killing. A blocking antibody against CD47 made the hypoimmunogenic CAR T cells susceptible to macrophage and NK cell killing, confirming the importance of CD47 overexpression to evade innate immune clearance. The use of CD47 blocking could additionally be envisioned as a safety strategy for our hypoimmunogenic CAR T cells. Neither isolated CD47 overexpression nor all three hypoimmune modifications or knockout of the TCR showed any effect on the cytotoxic potential of CAR+ T cells. Hypoimmune CAR+ T cells retain their antitumor activity in the Nalm-6 B cell leukemia model in vitro and clear leukemic cells in NSG mice across a range of tumor cell: CAR T cell ratios comparable to unmodified CAR T cells. These findings show that hypoimmunogenic CAR T cells are functionally immune evasive in allogeneic recipients with cytotoxic anti-tumor capacity and suggest they could provide universal CAR T cells that is able to persist without immunosuppression. Blocking CD47 could additionally serve as safety strategy for our hypoimmunogenic CAR T cells. Citation Format: Xiaomeng Hu, Mo Dao, Kathy White, Ryan Clarke, Sam Landry, Ron Basco, Corie Gattis, Eleonore Tham, Emily Luo, Andrew Tucker, Christopher Bandoro, Elaine Chu, Junmo Kim, Chi Young, William E. Dowdle, Edward J. Rebar, Terry J. Fry, Sonja Schrepfer. Overexpression of CD47 protects hypoimmune CAR T cells from innate immune cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB144.

Off-the-shelf CAR T cells may offer advantages over autologous strategies, including ease of manufacturing, improved quality control with avoidance of malignant contamination and T cell dysfunction as well as the ability to generate a final product from healthy T cells. While TCR editing can effectively prevent graft-versus-host reactions, the significant host-versus-graft immune response against histoincompatible T cells prevents the expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. The goal is to achieve improved rates of durable complete remissions by improving allogeneic CD19CAR persistence since it has been shown that autologous CAR T cells have greater durability over years than allogeneic CAR T cells (N Engl J Med. 2021;384(7):673-674).

Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.

SUPT4H1 is a transcription elongation factor that makes up part of the RNA polymerase II complex. Recent studies propose a selective role for SUPT4H1 in the transcription of repeat-containing DNA, the translated products of which contribute to neurodegenerative disorders such as C9orf72-amyotrophic lateral sclerosis. To investigate the potential of SUPT4H1 as a therapeutic target in repeat-associated neurodegeneration, we depleted SUPT4H1 by RNA interference to inhibit the function of the SUPT4H1/SUPT5H transcription elongation complex. Depletion of SUPT4H1 leads to a global reduction in all cellular RNA, highlighting the significant challenges that are associated with targeting this molecule for the treatment of human disease. Any requirement of SUPT4H1 for transcription of specific transcripts should be interpreted in the context of global modulatory effects on the transcriptome.

ABSTRACTHuman genetic variants are usually represented by four values with variable length: chromosome, position, reference and alternate alleles. Thereis no guarantee that these components are represented in a consistent way across different data sources, and processing variant-based data can be inefficient because four different comparison operations are needed for each variant, three of which are string comparisons. Working with strings, in contrast to numbers, poses extra challenges on computer memory allocation and data-representation. Existing variant identifiers do not typicallyrepresent every possible variant we may be interested in, nor they are directly reversible. To overcome these limitations,VariantKey, a novel reversible numerical encoding schema for human genetic variants, is presented here alongside a multi-language open-source software implementation (http://github.com/genomicspls/variantkey). VariantKey represents variants as single 64 bit numeric entities, while preserving the ability to be searched and sorted by chromosome and position. The individual components of short variants can be directly read back from the VariantKey, while long variants are supported with a fast lookup table.Highlights~100 compounds identified by high-content screen inhibit SGs in HEK293, NPCs and iPS-MNs.ALS-associated RBPs are recruited to SGs in an RNA-dependent mannerMolecules with planar moieties prevent recruitment of ALS-associated RBPs to SGsCompounds inhibit TDP-43 accumulation in SGs and inTARDBPmutant iPS-MNs.