Integration Site Research Articles

CD117 Antibody-Drug Conjugate Conditioning Allows Efficient Engraftment of Gene-Modified CD34+ Cells with Fertility Preservation in a Rhesus Gene Therapy Model

Hematopoietic stem cell (HSC) gene therapy is curative for multiple genetic diseases; however, it is limited due to toxicities from chemotherapy-based conditioning. To target HSCs directly, we developed an anti-human CD117 ( c-KIT)-targeted antibody-drug conjugate (CD117-ADC) conjugated to a DNA-binding payload that cross-reacted with non-human primates. To investigate the activity of CD117-ADC in non-human primates, we administered a single injection of ADC without HSC infusion, resulting in a >99% depletion of CD34+CD90+ bone marrow cells in cynomolgus macaques (n=2 of 3 in 0.1 mg/kg and n=3 in 0.3 mg/kg); similar to the ablation observed with 4 doses of myeloablative busulfan (Bu) (n=3). With escalating ADC doses (0.2-0.6 mg/kg), the optimal timing of HSC infusion (6 days in 0.2 or 0.3 mg/kg and 10 days in 0.4mg/kg) was determined in rhesus macaques (n=1 each). The animal dosed 0.6 mg/kg required euthanasia on day 14 due to irreversible hematopoietic suppression and severe liver toxicity. In our preliminary transplantation study, dosing 0.2 mg/kg ADC to rhesus macaques (n=2) resulted in bone marrow depletion and modest-level engraftment of gene-modified cells (vector copy number per cell (VCN) 0.05±0.01) (ASH 2019). Therefore, we evaluated higher single doses of CD117-ADC in rhesus macaques (0.3 mg/kg for ZL13 and ZJ62, and 0.4 mg/kg for H635 and H96G, n=4) and directly compared to Bu conditioning (5.5 mg/kg x 4 days, n=2). Mobilized rhesus CD34+ cells (ADC 6.4±2.9e6/kg vs. Bu 4.1±0.1e6/kg, n.s.) were transduced with a lentiviral vector for stable fetal hemoglobin (HbF) induction (Sci Transl Med. 2021). These cells ( in vitro VCN 10.1±3.8 vs. 10.2±7.3, n.s.) were transplanted into autologous animals after ADC or Bu conditioning. The time course of blood cell count suppression and recovery was similar between ADC and Bu animals, except for a strong reduction of platelet counts observed only with ADC (ASH 2021). Two months post-transplant, substantial levels of vector-containing granulocytes were observed long-term in 1 of 2 animals receiving 0.3 mg/kg ADC (ZJ62, VCN 0.16) and in both animals receiving 0.4 mg/kg (H635, VCN 0.46 and H96G, VCN 0.21) at a similar range to the two Bu animals (0.44±0.17, n.s.). Robust HbF induction was detected in three ADC animals with substantial VCN as measured by both HbF-positive cells (F-cells 8.5±1.8% vs. 13.7±5.8%, n.s.) and HPLC measurement of total HbF (8.0±2.9% vs. 11.1±5.2%, n.s.). In ZL13 (0.3 mg/kg ADC), lower gene marking (VCN 0.02) was observed, along with lower HbF induction (F-cells 1.0% and HbF amounts 0.9%). Importantly, ADC resulted in minimal off-target toxicity, unlike those associated with Bu (such as infertility, veno-occlusive disease, pulmonary dysfunction, mucositis, or seizures). High diversity of gene-modified cells (Simpson's diversity index >0.99) without any large clone (maximal integration site 0.3-3.7%) was observed by integration site analysis 1-year post-transplant with both ADC (0.3-0.4 mg/kg) and Bu. Infertility is a major complication of conventional myeloablative conditioning that limits the uptake of curative therapies; therefore, we investigated fertility in CD117-ADC animals. When monitored daily for menstrual bleeding, the menstrual cycles of all 4 female macaques (ZL13, ZJ62, H635, and H96G) were maintained post-ADC conditioning (0.92±0.11 per month) in contrast to our previously transplanted animals following myeloablative Bu (0.23±0.11, n=4, p<0.01) and total body irradiation (0.33±0.3, n=6, p<0.05). To confirm fertility, all transplanted animals post-ADC including females (n=4) and males (n=2) were housed for mating at a rhesus breeding colony. Two ADC females (ZL13 and H635) gave birth, and an ADC male (12U032) was paired with a pregnant female. Importantly, an ADC female (H635) was paired with an ADC male (13U047). In summary, a single-injection CD117-ADC allows for efficient engraftment of gene-modified CD34+ HSCs in a rhesus gene therapy model, achieving similar levels as the current clinical standard of care, myeloablative Bu conditioning. Robust HbF induction was also confirmed at the protein level in our rhesus gene therapy model with ADC conditioning. The transplanted animals with ADC maintained their fertility. Overall, these data indicate that an ADC-based targeted approach offers safer conditioning and could improve the risk-benefit profile in HSC gene therapy.

An In Vivo PiggyBac Insertional Mutagenesis Screen Reveals Oncogenic Lesions Cooperating with Myd88 L265P

Diffuse large B cell lymphoma (DLBCL) is the most common Non-Hodgkin lymphoma and originates from transformed germinal center-experienced B cells. Traditionally, DLBCL has been divided into two subtypes, depending on whether the transcriptional profile of the tumor relates to an activated B cell or a germinal center B cell (ABC and GCB DLBCL, Alizadeh et al., 2000). More recent efforts classified DLBCL cases based on their genetic aberrations and identified several clusters with distinct mutational profiles (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020). The MCD/C5 cluster is characterized by recurrent mutations in MYD88, PRDM1 and frequent amplifications of BCL2, amongst others. We recently showed that mice harboring a B cell-specific Myd88 L252P mutation (orthologous position of the human p.L265P mutation) develop B cell proliferation and occasional transformation into DLBCL (Knittel et al., 2016). Lymphomagenesis is further increased when Myd88 L252P is combined with BCL2 overexpression and a genetically engineered block in plasmacytic differentiation by loss of Prdm1 or overexpression of Spib (Flümann et al., 2021). To ask which further genes and pathways cooperate with Myd88 L252P in lymphomagenesis, we performed an in vivo piggyBac insertional mutagenesis screen. In this system, a conditionally expressed transposase mobilizes transposable elements. These elements can then reintegrate into the genome and either silence or drive the expression of genes, depending on the exact integration site and orientation of the transposon cassette (Rad et al., 2015). We crossed this piggyBac system onto a Myd88 L252P background to identify genes that cooperate with Myd88 mutations in lymphomagenesis. Mice harboring both the Myd88 mutant allele and the piggyBac system lived significantly shorter than controls harboring only the Myd88 mutation or just the piggyBac system. Myd88/piggyBac animals developed B220 +/CD138 - lymphomas and DNA isolated from these lesions allowed the detection of common transposon insertion sites. Among the genes significantly enriched for integrations, we observed known genetic drivers of human MCD/C5 DLBCL, such as PIM1 and ETV6. We also identified TBL1XR1 and SPIB as common insertion sites, as well as BCL2, BIM1 and BCL-XL, further validating our approach of engineering a plasma cell differentiation block as well as anti-apoptotic BCL2 overexpression on a Myd88-mutant background to model MCD DLBCL. Of note, the hits identified in this screen were significantly distinct from hits identified to drive Myc-driven B cell lymphomagenesis (Weber et al., 2019). Additionally, we identified several candidate genes that are not reported to be frequently genetically altered in DLBCL, however associated with processes relevant to B cell biology, particularly to B cell receptor signaling. As ‘B cell receptor signaling’ was a prominent term in our screen hits and CD79B ITAM mutations are an additional hallmark of MCD/C5 DLBCL (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020), we introduced a conditional Cd79b p.Y195H allele to our Cd19 Cre/wt;Myd88 cond.p.L252P/wt; Rosa26 LSL.BCL2-IRES-GFP/wt; Prdm1 fl/fl MCD/C5 DLBCL model. While we did not observe significant differences in the immunohistochemial and transcriptional phenotype of Cd79b WT and mutant lymphomas, Cd79b p.Y195H tumors showed increased B cell receptor (BCR) signaling activity, indicated by increased levels of phosphorylated SYK and PLCg2. Cd79b mutant lymphomas also showed an increased formation of cytoplasmic signaling complexes comprised of MYD88 and several components of the BCR pathway, including MALT1 and BTK. The formation of these complexes depended on active BTK, as treatment with the BTK inhibitor ibrutinib reduced complex formation to levels found in Cd79b WT lymphomas. Consequently, we investigated the effects of ibrutinib treatment in Cd79b-mutant and wildtype MCD DLBCL mouse models and found Cd79b-mutant lymphomas to be significantly more sensitive to ibrutinib treatment than their Cd79b WT counterparts.

Gene Therapy Expressing Platelet-Derived Factor VIII for Correction of Hemophilia Α with a History of Inhibitors

Introduction: Platelet targeted gene therapy with human factor VIII has established hemostasis in murine and canine models with severe hemophilia A withouteliciting inhibitory antibodies. Here-in we describe feasibility, safety and efficacy of the first subject treated on a first-in-human phase 1 trial that targets factor FVIII synthesis and storage within platelet α-granules for delivery at the site of vascular injury for hemophilia A (NCT03818763). Method: Eligible are severe hemophilia A patients ≥18 years with a history of inhibitors to factor VIII (≥0.6 Bethesda Units [BU]/ml). Following mobilization, autologous CD 34+ cells (≥6 x 10 6/kg) are collected and transduced with a lentiviral vector encoding the ITGA2B gene promoter for ectopic expression of human B-domain-deleted factor VIII within the megakaryocyte lineage. The transduced cells are infused after reduced intensity cytoreduction with fludarabine (120 mg/m 2) and melphalan (120 mg/m 2) followed by a washout period of ≥24 hours. The primary endpoints of feasibility and safety are defined as: 1) feasibility of the cell manufacturing procedure by the availability of ≥4 x 10 6/kg transduced clinical grade CD34+cells, cell viability ≥70% and undetectable microbial contamination and 2) safety defined as hematopoietic recovery ≤28 days of infusion and absence of ≥ grade 3 toxicity (CTCAE version 5.0). Outcome: Subject 1 is a 29-year-old male with severe hemophilia A who developed inhibitors to factor VIII (2.6 BU/ml) in his first year of life. Immune tolerance to factor VIII had been established with a non-detectable inhibitor titer at enrollment. Hemostatic prophylaxis was maintained with emicizumab weekly and recombinant factor VIII for breakthrough bleeding. His annualized bleeding rate was 16 in the 12-month period preceding infusion. The cell product was the result of 1 day of collection yielding 6.73 x 10 6 /kg total viable CD34+ cells post-transduction and vector copy number by qPCR of 1.16 copies/cell. Megakaryocyte factor VIII:C levels were 0.00 mU/10 6 before transduction and 101.32 mU/10 6 after transduction. The cell product satisfied release criteria. Neutrophil recovery (≥0.5 x 10 9/L) and platelet transfusion independence (≥50 x 10 9/L) was achieved 15 days post-infusion and sustained for >12 months. The duration of hospitalization was 21 days with no re-admissions. There was no breakthrough bleeding during collection and hospitalization. During and post-infusion there was no unexpected toxicity ≥ grade 1. He has not required immune suppression. Emicizumab was discontinued 3.6 months post-infusion after demonstration of whole blood vector copy number at 1 and 3-months. There has been no spontaneous bleeding or a need for “on demand” factor VIII after discontinuing emicizumab. Secondary outcomes are summarized in Table 1. No replication competent lentivirus was detected through month 12 post-infusion. Integration site analysis was carried out, and none of the samples tested through 12 months contained cell clones which exceeded 20% relative abundance. Transduced cells were highly polyclonal. Integration site analysis showed no enrichment of integration near cancer-associated genes in the cell product nor in whole blood cell lineages post-infusion. Conclusion: We report feasibility, safety and efficacy in the first subject with severe hemophilia A and a history of inhibitors who received lentiviral vector gene therapy directed to induce megakaryocytes to synthesize and store factor VIII within platelets. These findings extend the potential to treat severe hemophilia A patients who are not eligible for valoctcogene roxaoarvovec (AAV5-hFVIII-SQ).

Actionable Findings from an Unbiased Drug Screen for Novel Single Agent and Combination Therapies Against AML with Mecom Re-Arrangement

EVI1 (Ecotropic Viral Integration Site 1) is a zinc finger transcriptional regulator encoded from the MECOM (MDS1 and EVI1 Complex) locus at chromosome 3q26.2. Aberrant EVI1 overexpression is observed in approximately 10% of de novo AML, of which approximately half are due to chromosomal rearrangement involving either inv3(q21;q26.2) or t(3;3)(q21;q26.2), where the distal GATA2 hematopoietic enhancer is relocated and drives EVI1 expression. In addition to the resulting haploinsufficiency of GATA2, monosomy 7 and activating mutations in the RAS pathway and SF3B1 commonly co-occur in 3q26.2-rearranged (r) AML. AML with EVI1 overexpression generally have a stem-like phenotype, are refractory to current therapy options and exhibit poor overall survival. Among the targets of EVI1 are the ERG, MYC, KIT, Bcl-xL and MPL genes. GATA2 repression, coupled with increased EVI1 activity, promotes AML progression and an aggressive phenotype in 3q26.2-rearranged AML. Therefore, there is a glaring need to develop and test novel targeted therapies with improved efficacy in repressing EVI1 and improving survival of AML with EVI1 overexpression. To identify druggable vulnerabilities in 3q26.2-r AML, we conducted an unbiased high-throughput drug screen, utilizing a library of 2480 oncology-focused, mechanistically annotated drugs (referred as NCATS Mechanism Interrogation Plates or-MIPE 5.0) (Cancer Cell. 2021; 39:566-579), against seven AML cell lines, i.e., four with 3q26.2-r: UCSD-AML1, HNT-34, AML191 and AML194, and three non-3q26.2-r AML: SET-2, MV4-11 and OCI-AML3. The library exploits mechanistic redundancy by including multiple inhibitors against well-validated oncology targets, while simultaneously encompassing mechanistic diversity, altogether targeting more than 850 distinct mechanisms of action. We exploited this redundancy to identify druggable, target-level dependencies in 3q26.2-r AML, both in terms of absolute potency and relative activity as compared to other AML subtypes. BRD4 was identified as an absolute dependency in 3q26.2-r AMLs. This was consistent with previous reports that BET inhibitors (e.g., OTX015, mivebresib or ABBV-075 and JQ1) are effective against 3q26.2-r AML cell lines, patient-derived (PD) AML cells and PDX models. Comparison of 3q26.2-r versus other AML cell lines also identified XIAP, mTOR, PIK3CA and Bcl-xL as druggable vulnerabilities in 3q26.2-r AML. In follow-up experiments, XIAP/cIAPs inhibitors birinapant (10-1000 nM) or SM-164 (30-1000 nM), chosen based on the MIPE screen outcomes, induced significantly more dose-dependent apoptosis in 3q26.2-r versus the other AML cell lines. This was associated on Western analyses with decline in XIAP, c-IAP1/2, p-ERK1/2, MCL1 and Bcl-xL, but increased protein levels of cleaved caspase-3 and PARP in UCSD-AML1 and AML191 cells. Mivebresib (50-250 nM) or OTX015 (100-500 nM) also induced dose-dependent apoptosis, and reduced EVI1, c-Myc, c-Myb, XIAP, c-IAP1/2, CDK4/6 and Bcl-xL while increasing protein levels of HEXIM1 and cleaved PARP. Consistent with this, co-treatment with birinapant with mivebresib was synergistically lethal in UCSD-AML1 and AML191 cells, with Delta Synergy scores > 1.0 calculated by the ZIP method. Treatment with the dual mTOR/PIK3CA inhibitor NVP-BGT226 (1-30 nM) or navitoclax or Bcl-xL-specific BH3 mimetic A-1155463 also exerted lethality and synergistically induced apoptosis with mivebresib in AML cells with inv3/t(3;3). We previously reported that the TBL1/β-catenin antagonist tegavivint induced apoptosis in 3q26.2-r PD AML cells and PDX models. Co-treatment with birinapant and tegavivint also synergistically induced apoptosis in 3q26.2-r AML cells. Finally, in the xenograft model of AML194-Luc cells in NSG mice, monotherapy with birinapant (30 mg/kg/t.i.w, Intraperitoneal), vs vehicle control, significantly reduced the AML burden, without causing toxicity. Additionally, compared to each drug or vehicle control, co-treatment with birinapant and the BETi OTX015 (30 mg/kg/day, by oral gavage) was more effective in reducing AML burden in the xenograft model. These findings demonstrate promising preclinical activity of IAP protein inhibition against the cellular models of AML with inv3/t(3;3) with EVI1 overexpression, supporting the rationale to further evaluate in vivo efficacy of birinapant and/or BETi-based combinations against this AML sub-type.

Feasibility study on energy harvesting with thermoelectric generators in a photovoltaic-ground source heat pump system

Thermoelectric generators (TEGs) harness temperature differences to produce electricity and hold promise for diverse industrial applications. However, their limited conversion efficiency casts doubt on their role in achieving energy independence. This study introduces an advanced technique that exploits temperature gradients in water pipes, utilizing supplementary TEGs to augment power generation. This method maintains a stable temperature gradient for TEG operation. Additionally, TEG power output can be efficiently modulated via flow control. In the feasibility evaluation for residential settings, the temperature fluctuations across each system unit were analyzed. In the active system, the chosen sites for TEG integration were units equipped to manage heat transfer using working fluids. The inlet and outlet temperatures were calculated for photovoltaic-thermal (PVT) systems, ground heat exchangers (GHEs), and heat storage tanks (HSTs). The electricity produced by the TEGs was benchmarked against their conversion efficiency, zT. The results indicated that the TEGs yielded 10.95 kWh of electricity when systematically implemented in each unit. To realize a zero-energy building, an area of 64.5 m2 per unit is necessitated for TEG deployment, given a zT value of 1.

Genomic and Phenotypic Characterization of Shiga Toxin-Producing Escherichia albertii Strains Isolated from Wild Birds in a Major Agricultural Region in California.

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

Characteristics of HPV integration in cervical adenocarcinoma and squamous carcinoma

PurposeHPV integration usually occurs in HPV-related cancer, and is the main cause of cancer. But the carcinogenic mechanism of HPV integration is unclear. The study aims to provide a theoretical basis for understanding the pathogenesis of cervical adenocarcinoma (AC) and cervical squamous carcinoma (SCC).MethodsWe used HPV capture sequencing to obtain HPV integration sites in AC and SCC, and analyzed cytobands, distribution of genetic and genomic elements, identified integration hotspot genes, clinicopathological parameters, breakpoints of HPV16 and performed pathway analysis. Then we conducted immunohistochemical (IHC) assay to preliminarily verify the expression of most frequently integrated genes in AC, STARD3 and ERBB2.ResultsThe results revealed that the most frequently observed integrated cytoband was 17q12 in AC and 21p11.2 in SCC, respectively. The breakpoints in both AC and SCC were more tended to occur within gene regions, compared to intergenetic regions. Compared to SCC samples, AC samples had a higher prevalence of genomic elements. In AC, HPV integration has no significantly difference with clinicopathological parameters, but in SCC integration correlated with differentiation (P < 0.05). Breakpoints of HPV in SCC located in LCR more frequently compared to AC, which destroyed the activation of promoter p97. Hotspot genes of HPV integration were STARD3 and ERBB2 in AC, and RNA45S rDNA and MIR3648-1 in SCC, respectively. Meanwhile, we preliminarily proved that the expression of STARD3 and ERBB2, the most frequently integrated genes, would increase after integration.ConclusionThese results suggested that HPV may utilize the powerful hosts’ promoters to express viral oncogenes and overexpression of viral oncogenes plays a significant role in the carcinogenesis of SCC. In AC, HPV integration may affect hosts’ oncogenes, and the dysregulation of oncogenes may primarily contribute to progression of AC.

Viral and host mediators of non-suppressible HIV-1 viremia

Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as ‘producer proviruses’, and those that did not as ‘non-producer proviruses’. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4+ T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8+ T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence.

Biophysical model for DNA mutations induced by retroviral genome insertion based on the probability density function of mutation distribution

Research into the biophysical properties of deoxyribonucleic acid (DNA) and the mechanisms underlying genetic mutations has undergone marked advancements. The intriguing nature of mutations resulting from retroviral DNA insertion has garnered considerable attention. Whether these mutations are random or region-specific, the distribution patterns of mutation sites have been the focus of numerous research endeavours. This mutation mechanism originates from interactions between host DNA and the pre-integration complex (PIC), comprising retroviral DNA and an integrase enzyme that facilitates its incorporation into the host DNA. Our study focused on the Zfp521 gene locus, recognised for its pronounced susceptibility to insertional mutations, particularly around unique palindromic sequences. We employed two biophysical models to predict mutation distribution within a range of 50 base pairs centred on these sequences. The first is a probabilistic collision model emphasising PIC and target DNA interactions. The second model is a DNA diffraction lattice, where the PIC behaves according to probability density. Although both models adeptly illuminated the probability distributions of target sites, the second model was more successful in predicting the PIC integration sites based on DNA biophysical properties. This highlights the pivotal role of intricate interactions between the PIC and target DNA, suggesting that mutations can be predicted in a stochastic manner.

Generation of hepatitis C virus–resistant liver cells by genome editing–mediated stable expression of RNA aptamer

Hepatitis C virus (HCV) infections frequently recur after liver transplantation in patients with HCV-related liver diseases. About 30% of these patients progress to cirrhosis within five years after surgery. In this study, we proposed an effective therapeutic strategy to overcome the recurrence of HCV. CRISPR/Cas9 was used to insert an expression cassette encoding an RNA aptamer targeting HCV NS5B replicase as an anti-HCV agent into adeno-associated virus integration site 1 (AAVS1), known as a “safe harbor”, in a hepatocellular carcinoma cell line to confer resistance to HCV. The RNA aptamer expression system based on a dihydrofolate reductase (DFHR) minigene was precisely knocked-in into AAVS1, leading to the stable expression of aptamer RNA in the developed cell line. HCV replication was effectively inhibited at both the RNA and protein levels in cells transfected with HCV RNA or infected with HCV. RNA immunoprecipitation and competition experiments strongly suggested that this HCV inhibition was due to the RNA aptamer-mediated sequestration of HCV NS5B. No off-target insertion of the RNA aptamer expression construct was observed. The findings suggest that HCV-resistant liver cells produced by genome editing technology could be utilized as a new alternative in the development of a treatment for HCV-induced liver diseases.

Abstract 17416: Epigenetic Regulation of Fibroblast Fate Transition in Cardiac Wound Healing

Background: Fibroblast transitioning between physiological and pathological states can define cardiac wound healing ability of the heart in response to stress. Preventing fibroblast activation and differentiation into myofibroblast formation can attenuate cardiac fibrosis and adverse cardiac remodeling. Changes in chromatin structure and accessibility are one of the essential drivers of gene expression/repression involved in myocardial injury. One such driver of chromatin remodeling is the group of polycomb proteins that alter expression of genes involved in cardiac wound healing including B cell specific Moloney murine leukemia virus integration site 1 (Bmi1), a component of the polycomb repressive complex 1 (PRC1). Bmi1 have been linked to self-renewal, proliferation, quiescence, and lineage commitment in many organs however, how Bmi1 can mediate fibroblasts states and mediate wound healing after cardiac injury is not understood. Hypothesis: Bmi1 mediates myofibroblast activation and transition, scar maturation, stability, and fibrotic responses by regulating histone marks after injury. Methods and Results: To establish the role of Bmi1 in cardiac wound healing, we developed an inducible knock out mouse model for Bmi1, which showed decrease in cardiac structure and function with maladaptive remodeling following myocardial injury. Bmi1 knockout (KO) mice showed significant decrease in ejection fraction and fractional shortening measured by echocardiography after cardiac injury. Concurrently, this significant decrease in heart function was associated with increased fibrosis and DNA damage analyzed using immunohistochemistry. Additionally, we found that the adult cardiac fibroblasts, isolated from the Bmi1 KO mice have increased expression of fibrotic genes including TGFβ, Acta2, and collagen using RTPCR analysis. Using single cell sequencing RNA and ATAC sequencing and cellular staining assays, we found significant changes in fibrotic signaling pathways of TGFβ. We found SMAD3 chromatin accessibility was increased with the loss of Bmi1 with the decrease in H2AK119Ub. Conclusion: Bmi1 mediates wound healing ability by regulating cardiac fibroblast transition after cardiac injury.

When the Trial Ends: The Case for Post-Trial Provisions in Clinical Psychedelic Research

The ethical value—and to some scholars, necessity—of providing trial patients with post-trial access (PTA) to an investigational drug has been subject to significant attention in the field of research ethics. Although no consensus has emerged, it seems clear that, in some trial contexts, various factors make PTA particularly appropriate. We outline the atypical aspects of psychedelic clinical trials that support the case for introducing the provision of PTA within research in this field, including the broader legal status of psychedelics, the nature of the researcher-therapist/participant relationship, and the extended time-frame of the full therapeutic process. As is increasingly understood, the efficacy of psychedelic-assisted psychotherapy is driven as much by extrapharmacological elements and the cultural therapeutic container as by the drug itself. As such, we also advocate for a refocusing of attention from post-trial access to a broader concept encompassing other elements of post-trial care. We provide an overview of some of the potential post-trial care provisions that may be appropriate in psychedelic clinical trials. Although the World Medical Association’s Declaration of Helsinki calls on researchers, sponsors, and governments to make provisions for post-trial access, such provision may feel impracticable or out-of-reach within psychedelic trials that are already constrained by a high resource demand and significant bureaucratic burden. We show how conceiving of post-trial provision as an integral site of the research process, and an appropriate destination for research funding, will serve to develop the infrastructure necessary for the post-legalisation psychedelic medicine ecosystem.

The analysis of HPV integration sites based on nanopore sequencing and the profiling changes along the course of photodynamic therapy

ObjectiveTo detect the HPV genotype and integration sites in patients with high-risk HPV infection at different stages of photodynamic therapy using nanopore technology and to evaluate the treatment effect.MethodsFour patients with HPV infection were selected and subjected to photodynamic therapy, and cervical exfoliated cell was sampled at before treatment, after three courses of treatment and six courses of treatment, their viral abundance and insertion sites were analyzed by nanopore technology, and pathological examinations were performed before and after treatment. In this study, we developed a novel assay that combined viral sequence enrichment and Nanopore sequencing for identification of HPV genotype and integration sites at once. The assay has obvious advantages over qPCR or NGS-based methods, as it has better sensitivity after viral sequences enrichment and can generate long-reads (kb to Mb) for better detection rate of structure variations, moreover, fast turn-around time for real-time viral sequencing and analysis.ResultsThe pathological grade was reduced in all four patients after photodynamic therapy. Virus has been cleared in two cases after treatment, the virus amount reduced after treatment but not completely cleared in one case, and two type viruses were cleared and one type virus persisted after treatment in the last patient with multiple infection. Viral abundance and the number of integration sites were positively correlated. Gene enrichment analysis showed complete viral clearance in 1 patient and 3 patients required follow-up.ConclusionNanopore sequencing can effectively monitor the abundance of HPV viruses and integration sites to show the presence status of viruses, and combined with the results of gene enrichment analysis, the treatment effect can be dynamically assessed.

CAR+ T-Cell Lymphoma Post Ciltacabtagene Autoleucel Therapy for Relapsed Refractory Multiple Myeloma

Introduction:Rareevents of T-cell lymphoma (TCL) derived from CAR-T cells (2 cases) have been reported in patients receiving nonviral piggyBac transposon-based CAR-T therapy (Micklethwaite et al, Blood, 2021). Ciltacabtagene autoleucel (cilta-cel) is an anti-BCMA CAR-T therapy produced via conventional lentiviral transduction. In the randomized, phase 3 CARTITUDE-4 study (NCT04181827), cilta-cel significantly improved PFS (HR=0.26) vs standard of care in lenalidomide-refractory patients with multiple myeloma and 1-3 prior lines of therapy. We present the clinicogenomic characterization of a CARTITUDE-4 patient who developed a CAR+ TCL post cilta-cel. Methods: Diagnostic and staging workup included biopsy analyses and FDG-PET scan. Presence of CAR+ cells in lymph node biopsy (LNB) was assessed by quantitative polymerase chain reaction (qPCR), in situ hybridization (ISH), and immunohistochemistry (IHC). Whole genome sequencing (WGS), transcriptome sequencing, whole exome sequencing (WES), T-cell receptor (TCR) sequencing, and genome-wide CAR integration analyses were conducted. Results:A 51 y/o male patient received cilta-cel; CAR+ T cells in blood peaked 14 d post-infusion (77 cells/μL) and decreased to 3 cells/μL at d 92 post-infusion, when he achieved stringent complete response (sCR) and MRD negativity at 10 -5. At 5 mo post-infusion, a relatively rapidly growing erythematous nasofacial plaque developed. TCL was diagnosed based on facial lesion biopsy showing an infiltrate of atypical T cells positive for CD2 and CD3 but negative for CD4, CD8, CD7, CD56, ALK, EBER-ISH, TdT, CD30, and cytotoxic T cell markers. FDG-PET showed bilateral FDG-avid cervical lymphadenopathy, with similar T cell infiltrate in submandibular LNB. qPCR and ISH/IHC revealed 90-100% of LNB cells to be CAR+. At d 162 post-infusion (after TCL diagnosis but before TCL-directed chemotherapy) CAR+ T cells in blood had re-expanded, independent of BCMA antigen, to 378 cells/μL. CAR integration analysis of LNB revealed a dominant insertion into the 3′UTR of PBX2 (91.1% of total reads from all integration sites), suggesting tumor monoclonality. TCR sequencing (1.8×10 -6 sensitivity) of LNB showed a monoclonal sequence in 91% of all T cells. Analysis of the drug product revealed the same unique TCR sequence from the monoclone at low frequency (~2×10 -6), suggesting the presence of this clone in apheresis material. WGS showed low overall mutational burden in the LNB (1.26 mutations/megabase) with 37 coding and 3286 non-coding variants, including 2 predicted loss-of-function TET2 mutations; a PTPRB truncation; and a focal duplication involving the 5′UTR to intron 16 of NFKB2. No dominant mutational signatures or gross copy number changes were observed. WES of the LNB indicated that a TET2 mutation (p.H1416R), which was not due to CAR insertion, was heterozygous and likely clonal. Targeted sequencing (0.5% sensitivity) of stored CD34+ cells and bone marrow aspirate collected from the patient 2 y earlier showed no abnormalities. Germline samples revealed the presence of a heterozygous JAK3 variant (p.V722I) that has been described as an activating variant, implicated in TCL, and detected in germline samples from patients with antigen-induced TCL (Blombery et al, Haematologica, 2016). The patient received CHOEP-21 (cyclophosphamide-doxorubicin-vincristine-prednisone-etoposide) and achieved metabolic CR but relapsed soon after treatment was stopped. Subsequent treatment with gemcitabine-dexamethasone-cisplatin-alemtuzumab was followed by consolidation with fludarabine plus melphalan and matched allogeneic stem cell transplant. He relapsed with cutaneous disease within 3 mo. Conclusions: To our knowledge, this is the first case of CAR+ TCL occurring after infusion of a CAR-T therapy produced via lentiviral transduction (cilta-cel). This rare malignancy was potentially driven by genetic mutations (e.g., TET2, NFKB2, PTPRB and/or JAK3), some of which may have existed in the form of a clone with malignant potential before cilta-cel manufacturing (e.g., TET2 p.H1416R and JAK3 p.V722I variants). A potential contributory role of the CAR insertion in the 3′ untranslated region of PBX2 to TCL development remains unclear and cannot be excluded at this time. Further investigation is needed to elucidate the differential contributions of these genomic factors to the etiology of this TCL case.

Integrated Genomic Analysis Identifies a Downstream Regulatory Element Oncogenically Activating Hematopoietically Expressed Homeobox ( HHEX) in ETP-ALL

Hematopoietically-expressed Homeobox ( HHEX) is required for the maintenance of hematopoietic stem cells (HSPCs) and common lymphoid progenitor cells. HHEX is also the second most frequent integration site in retroviral insertional mutagenesis screens of leukemias and lymphomas arising in AKXD recombinant inbred mouse strains, implying that it is an important oncogene. Enforced expression of HHEX induces T-cell acute lymphoblastic leukemias (T-ALL) in murine bone marrow transplantation models. In human studies, HHEXmRNA expression is upregulated in human T-ALL studies, especially in Early T-cell Precursor (ETP-) ALL subtypes where it is concordantly expressed with LMO2. The HHEX locus is not rearranged in ETP-ALL where it is upregulated so the mechanism of HHEX activation is not clear. We analyzed T-ALL induced by retroviral mutagenesis and found an intergenic site of frequent integration, 65kb 3‘ of the Hhex coding exons. Integrations clustered within a 3 kb genomic area that is also an open chromatin region (OCR) by ATAC-seq analysis and that induced Hhex upregulation. The syntenic human region was highly enriched for H3K27 acetylation and for occupancy by multiple transcription factors, notably GATA1 and TAL1, per ENCODE. Our prior data had shown that HHEX was a downstream target of the LMO2 complex comprised of GATA1/2, TAL1 or LYL1, and nucleated by the scaffolding protein, LIM domain binding protein 1 (LDB1). We had previously shown by ChIP-PCR that the LMO2 complex was bound to the intron 1 enhancer. Thus, we performed ChIP-exonuclease analysis of LDB1 in the human ETP-ALL model cell line, LOUCY. We confirmed its occupancy at the intron 1 enhancer but we also observed LDB1 occupancy 68kb 3‘ of the HHEX coding exons, in the exact region syntenic to the murine common insertion sites. ChIP-exo analysis allowed us to pinpoint a core element with composite GATA/E box sites that was highly conserved across multiple mammalian species. LDB1 occupancy at intron 1 and at +68kb of the HHEX locus was reminiscent of LDB1's occupancy at the beta globin locus where LDB1's occupancy and homodimerization mediates chromatin looping between composite E box/GATA sites in the locus control region (LCR) and the beta globin proximal promoters. To test whether a similar interaction was occurring at the HHEX locus, we analyzed virtual 4C (chromatin conformation capture) data. We confirmed looping between the +68kb element and the proximal promoter of HHEX in human ETP-ALL primary samples and in human HSPCs. The experiments showed that looping occurs in certain developmental contexts in HSPCs and is recapitulated in ETP-ALL and mediated by the LMO2/LDB1 protein complex. HHEX is a downstream oncogene of the LMO2/LDB1 complex and is activated through this distal +68kb regulatory element. Our studies raise the possibility that dissection of this regulatory element and interference with chromatin looping are potential therapeutic mechanisms that could disrupt oncogene expression.

Identification of ZFP91 As the Master Transcription Factor for EVI1 Gene Expression

Ecotropic viral integration site 1 (EVI1) is essential for hematopoiesis, particularly for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is widely known to be an independent poor prognostic factor in acute myeloid leukemia (AML). Previous studies have shown that enhancer hijacking or gene fusion caused by chromosomal translocation or inversion involving 3q26 leads to EVI1 overexpression. Intriguingly, however, aberrant EVI1 expression is seen in many cases of AML without 3q26 rearrangement. The prognosis of these cases is similarly dismal, but the mechanism that causes aberrant EVI1 expression in the absence of 3q26 chromosomal abnormalities is largely unknown. To identify key regulatory factors essential for EVI1 expression in AML, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout screen. We knocked in the green fluorescent protein (GFP) coding sequence at the EVI1 locus in the human myeloid leukemia cell line K562, which expresses high levels of EVI1 without a 3q26 chromosomal aberration. After transduction of a genome-wide CRISPR/Cas9 knockout library, transduced cells were separated into GFP High and GFP Low subpopulations by flow cytometry. By sequencing the uniquely attached barcodes, we estimated the effect of gene knockout for each gene. Genes counted more frequently in the GFP Low fraction were listed as candidates for activators of EVI1. The top 13 genes, including three apparent positive controls such as EVI1 and two ribosome-related genes, had a false discovery rate (FDR) of less than 1%. We validated these candidate genes in K562 and two other EVI1-overexpressing AML cell lines by transducing individual single guide RNAs targeting these genes. These analyses identified zinc finger protein 91 (ZFP91) as a potential regulatory factor that significantly upregulates EVI1 expression common to the three independent myeloid leukemia cell lines. ZFP91 is an atypical E3 ubiquitin ligase, a DNA-binding protein that functions as a transcription factor. ZFP91 promotes cell growth and inhibits apoptosis in AML and has been shown in previous studies to activate multiple oncogenic pathways, including nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and hypoxia-inducible factor-1α (HIF-1α). We demonstrated that knockout of ZFP91 in K562 substantially reduced EVI1 protein expression to undetectable levels by Western blotting (Figure 1). EVI1 mRNA expression also decreased dramatically after ZFP91 knockout. These results suggest that ZFP91 may play an indispensable role in EVI1 transcription. We performed chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) targeting the EVI1 promoter in K562 and demonstrated that ZFP91 knockout clones showed approximately 10-fold weaker signals for active histone modifications (H3K4me3 and H3K27ac) at the EVI1 promoter (Figure 2). In addition, chromatin immunoprecipitation sequencing (ChIP-seq) for ZFP91 in K562 revealed the presence of a distinct peak as well as the ZFP91-specific motif sequence within the EVI1 promoter region. These data strongly suggest that ZFP91 functions as an essential transcription factor for EVI1 gene overexpression in some EVI1-associated myeloid leukemias. We confirmed that ZFP91 knockout in K562 inhibited cell proliferation, which is consistent with previous studies in other AML cell lines. Based on these results, we believe that ZFP91 has the potential to serve as a therapeutic target in EVI1-associated AML. To conclude, we have identified the transcription factor ZFP91 as a novel prominent activator of EVI1 expression in some EVI1-associated AML. Our study suggests that ZFP91 may be the key to unraveling the pathogenesis of EVI1-associated AML and an attractive therapeutic target in EVI1-associated AML.

InCliniGene enables high-throughput andcomprehensive in vivo clonal tracking toward clinical genomics data integration.

High-throughput clonal tracking in patients under hematopoietic stem cell gene therapy with integrating vector is instrumental in assessing bio-safety and efficacy. Monitoring the fate of millions of transplanted clones and their progeny across differentiation and proliferation over time leverages the identification of the vector integration sites, used as surrogates of clonal identity. Although γ-tracking retroviral insertion sites (γ-TRIS) is the state-of-the-art algorithm for clonal identification, the computational drawbacks in the tracking algorithm, based on a combinatorial all-versus-all strategy, limit its use in clinical studies with several thousands of samples per patient. We developed the first clonal tracking graph database, InCliniGene (https://github.com/calabrialab/InCliniGene), that imports the output files of γ-TRIS and generates the graph of clones (nodes) connected by arches if two nodes share common genomic features as defined by the γ-TRIS rules. Embedding both clonal data and their connections in the graph, InCliniGene can track all clones longitudinally over samples through data queries that fully explore the graph. This approach resulted in being highly accurate and scalable. We validated InCliniGene using an in vitro dataset, specifically designed to mimic clinical cases, and tested the accuracy and precision. InCliniGene allows extensive use of γ-TRIS in large gene therapy clinical applications and naturally realizes the full data integration of molecular and genomics data, clinical and treatment measurements and genomic annotations. Further extensions of InCliniGene with data federation and with application programming interface will support data mining toward precision, personalized and predictive medicine in gene therapy. Database URL: https://github.com/calabrialab/InCliniGene.

The radicality and cultural significance of the sweats in Trinidad and Tobago

ABSTRACT The sweat, meaning to sweat, is a moniker attached to prearranged but unorganised and informal sport and physical activity within local communities in Trinidad and Tobago. Explained as a culturally significant phenomenon, the sweat is a space of community cohesion and radical questioning through its resident attendant ole talk where the critical deconstruction of social reality readily occurs. Using Paolo Freire’s pedagogical ideas of problem-posing dialogue, this ethnographic paper, explores three main areas. Those being, the sweats and ole talk; the radicality of the sweats displayed in individual agency against the structured restrictions of formal sports participation; and the sweats as a site for cross-cultural integration and interaction in an otherwise ethnically diverse country. Through the experiences of eight individuals associated to the sweats, Freirean ideas of love, radicality and hopefulness are prominent in the description of the sweats. To this end, this paper adds to the growing body of literature on informal sport participation as a site to negotiate and reconcile differences in local communities, as well as a site for social and sport-for-development.

Effect and mechanism of Jiming Powder on myocardial fibrosis in mice with myocardial infarction

Jiming Powder is a traditional ancient prescription with good therapeutic effect in the treatment of heart failure, but its mechanism lacks further exploration. In this study, a mouse model of coronary artery ligation was used to evaluate the effect and mechanism of Jiming Powder on myocardial fibrosis in mice with myocardial infarction. The study constructed a mouse model of heart failure after myocardial infarction using the method of left anterior descending coronary artery ligation. The efficacy of Jiming Powder was evaluated from multiple angles, including ultrasound imaging, hematoxylin-eosin(HE) staining, Masson staining, Sirius Red staining, and serum myocardial enzyme spectrum detection. Western blot analysis was performed to detect key proteins involved in ventricular remodeling, including transforming growth factor-β1(TGF-β1), α-smooth muscle actin(α-SMA), wingless-type MMTV integration site family member 3a(Wnt3a), β-catenin, matrix metallopeptidase 2(MMP2), matrix metallopeptidase 3(MMP3), TIMP metallopeptidase inhibitor 1(TIMP1), and TIMP metallopeptidase inhibitor 2(TIMP2). The results showed that compared with the model group, the high and low-dose Jiming Powder significantly reduced the left ventricular internal diameter in systole(LVID;s) and diastole(LVID;d), increased the left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), effectively improved cardiac function in mice after myocardial infarction, and effectively reduced the levels of myocardial injury markers such as creatine kinase(CK), creatine kinase isoenzyme(CK-MB), and lactic dehydrogenase(LDH), thus protecting ischemic myocardium. HE staining showed that Jiming Powder could attenuate myocardial inflammatory cell infiltration after myocardial infarction. Masson and Sirius Red staining demonstrated that Jiming Powder effectively inhibited myocardial fibrosis, reduced the collagen Ⅰ/Ⅲ ratio in myocardial tissues, and improved collagen remodeling after myocardial infarction. Western blot results showed that Jiming Powder reduced the expression of TGF-β1, α-SMA, Wnt3a, and β-catenin, decreased the levels of MMP2, MMP3, and TIMP2, and increased the level of TIMP1, suggesting its role in inhibiting cardiac fibroblast transformation, reducing extracellular matrix metabolism in myocardial cells, and lowering collagen Ⅰ and α-SMA content, thus exerting an anti-myocardial fibrosis effect after myocardial infarction. This study revealed the role of Jiming Powder in improving ventricular remodeling and treating myocardial infarction, laying the foundation for further research on the pharmacological effect of Jiming Powder.

Modulating cardiac physiology in engineered heart tissue with the bidirectional optogenetic tool BiPOLES

Optogenetic actuators are rapidly advancing tools used to control physiology in excitable cells, such as neurons and cardiomyocytes. In neuroscience, these tools have been used to either excite or inhibit neuronal activity. Cell type–targeted actuators have allowed to study the function of distinct cell populations. Whereas the first described cation channelrhodopsins allowed to excite specific neuronal cell populations, anion channelrhodopsins were used to inhibit neuronal activity. To allow for simultaneous excitation and inhibition, opsin combinations with low spectral overlap were introduced. BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing) is a bidirectional optogenetic tool consisting of the anion channel Guillardia theta anion-conducting channelrhodopsin 2 (GtACR2 with a blue excitation spectrum and the red-shifted cation channel Chrimson. Here, we studied the effects of BiPOLES activation in cardiomyocytes. For this, we knocked in BiPOLES into the adeno-associated virus integration site 1 (AAVS1) locus of human-induced pluripotent stem cells (hiPSC), subjected these to cardiac differentiation, and generated BiPOLES expressing engineered heart tissue (EHT) for physiological characterization. Continuous light application activating either GtACR2 or Chrimson resulted in cardiomyocyte depolarization and thus stopped EHT contractility. In contrast, short light pulses, with red as well as with blue light, triggered action potentials (AP) up to a rate of 240 bpm. In summary, we demonstrate that cation, as well as anion channelrhodopsins, can be used to activate stem cell–derived cardiomyocytes with pulsed photostimulation but also to silence cardiac contractility with prolonged photostimulation.