ALBERT

Description: An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

Description: Key PointsRapamycin significantly enhances lentiviral vector gene delivery to hematopoietic stem cells while preserving engraftment potential. Rapamycin-mediated transduction enhancement is not accompanied by alterations in lentiviral integration profile.

Description: To evaluate levels of coagulation /anticoagulation factors and distribution of their gene polymorphisms in patients with venous thromboembolism(VTE) and acute myocardial infarction(AMI). Of 95 VTE, 95 AMI patients and 95 normal controls were be studied. The results showed that the levels of fibrinogen and D-Dimer, the activies of FII, FV and FVIII were remarkably higher in patient with VTE than those in healthy group, and the activities of FIX, PC and AT were of no significant difference between VTE group and healthy group. The mean levels of fibrinogen, D-Dimer, FV:C and FVIII:C were significantly higher in AMI group than that in controls; while, no significant difference of FII:C, FIX:C, PC:A and AT:A between AMI patients and the controls. In controls, -148CT + -148TT was associated with higher fibrinogen concentrations, the genotypes of other gene polymorphisms were not associated with levels of coagulation /anticoagulation factors. Clear difference were observed for the T allele frequencies of -148C/T and distribution frequencies of FII A19911G were found between VTE group and normal group. there was significant difference of -1476TT genotype distributions and T allele frequencies in -1476A/T between VTE and control subjects. There was significant difference of AC+CC genotype distribution in 1298 A/C polymorphisms between AMI and control, and it was so with the GA genotype in 1793 G/A polymorphisms. But neither genotype frequencies nor allele frequencies of other polymorphisms were found to be no of significant difference between VTE /AMI group and control group. This study proved the importance of combined functions of several coagulant factors in causing VTE and AMI. The carriage of the C148T mutation, FII 20210G/A and -1476A/T polymorphism might be associated with an increased risk of VTE, while the MTHFR 1298A/C and 1793G/A polymorphisms may be related to the development of AMI. Only some of joint occurrence of above polymorphisms might increase the occurrence rate of VTE or AMI.

Description: Introduction: One of the main complications of adoptive T cell therapy (ACT) is the en-masse activation of tumor-reactive T cells inducing a large release of cytokines followed by activation of other immune cells leading to adverse events. These are classified as a cytokine release syndrome (CRS) or neurotoxicity described as a CAR T Related Encephalopathy Syndrome (CRES). Several biomarkers have been associated with CRS and/or neurotoxicity such as LDH, ferritin and CRP. Cytokines have also been associated with CRS and and/or CRES, but present approaches rely on retrospective study of collected biomarkers. Here, we report the results of cytokine analysis using a point of care (POC) device to predict immune-related toxicities in patients with relapsed/refractory (R/R)DLBCL treated with axicabtagene ciloleucel (axi-cel). Methods: Patients with R/R DLBCL treated with commercial axi-cel were included in this study. Baseline serum samples were collected prior to lymphodepleting chemotherapy and then daily during hospitalization. To select which cytokines to monitor, we retrospectively analyzed 38 serum cytokines in a cohort of 53 patients with R/R B cell acute lymphoblastic leukemia (B-ALL) who were treated with 19-28z CAR T cells. The patients were divided into those requiring treatment with tocilizumab and/or steroids versus those who did not require treatment. We observed several cytokines, including IL-2, IL-6, IL-15 and IFNg, which were significantly elevated in patients with CRS and/or CRES requiring treatment (Figure 1a). Based on this analysis and results of published studies, eight serum proteins were selected in our study including IL-1b, IL-2, IL-6, IL-15, IFNg, TNFa, and angiopoietin-1 &2. We monitored these proteins using a POC device that allows for rapid daily monitoring with a turnaround time of two hours. We established that the results from the POC device strongly correlate with a current gold standard device(Luminex), which has a typical two day turn around time. CRS and CRES were prospectively graded using revised Lee criteria (Lee et al Blood 2014) and the CARTOX group (Neelapu et al. NRCO 2017) respectively by an experienced clinical team and confirmed by chart review retrospectively. Results: A total of 20 patients with R/R DLBCL treated with commercial axi-cel were identified. Median age 64 years ( range 43-73) with 80% male.In our cohort, grades 1-3 CRS were observed in 45%, 40% and 5% respectively. There were no observed grade 0 or grade 4 CRS. There were two patients (10%) who died in the setting of severe toxicity. Patients with grade 5 CRS had higher levels of IL-6 and angiopoietin 2/angiopoietin 1 ratio at day one, which correlated with severity of toxicity r=0.52 (p= 0.039) , and r=0.53 (p=0.033) respectively (Fig. 1b). Furthermore, patients with high grades CRS had elevated levels of IL-15 at day seven (r=0.83, p=0.006). The majority of patients (55%) had grade 1-2 CRES.There were no significant correlations between serum cytokine levels and CRES or between those who required tocilizumab/steroids vs. those who did not, likely due to the small sample size. In select cases, daily monitoring of cytokines using the POC device provided clinical insight that wasn't evident from standard biomarkers. For example, one patient who developed delayed CRS had high serum levels of IL-6 but did not have elevated levels of CRP(Fig.1c). Discussion: In this analysis of 20 patients, we observed a correlation between severe CRS and elevated serum cytokine levels of IL-6 and angiopoietin 2/angiopoietin 1 ratio at day one suggesting that these biomarkers may be utilized to predict severe toxicity in patients treated with ACT. While this study is limited by small sample size, our observations correlate with previously published biomarkers data in patients enrolled in clinical trials. To our knowledge this is the first reported cytokine data using commercial axi-cel. Monitoring of cytokines using a POC device is feasible and will be useful clinically. High risk patients may be identified early and help guide intervention in real time, for example day one elevated IL-6 levels might inform earlier use of tocilizumab. We continue to enroll patients to validate cytokines as predictive biomarkers with the goal of informing the development of preventative strategies to mitigate CAR T cell therapy immune related adverse events. Disclosures Locke: Cellular BioMedicine Group Inc.: Consultancy; Kite Pharma: Other: Scientific Advisor; Novartis Pharmaceuticals: Other: Scientific Advisor. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Park:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Kite Pharma: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Shire: Consultancy. Davila:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: Of patients receiving CD19 CAR T cell therapy for large B cell lymphoma (LBCL), approximately 1 in 10 experience severe cytokine release syndrome (CRS) and 1 in 3 experience severe neurotoxicity. While CAR T cells trigger the onset of these toxicities, CRS and neurotoxicity are thought to occur as a consequence of activated myeloid cells amplifying cytokine and catecholamine release, thereby stimulating inflammation both systemically and at the blood-brain barrier. However, patient and tumor-related factors that account for differences in the amount of toxicity remain poorly understood. Methods: Serum cytokine levels were measured on an ELLA point of care device prior to lymphodepleting chemotherapy and throughout inpatient treatment with CD19 CAR T cell therapy (axicabtagene ciloleucel) for LBCL. Catecholamine levels were measured as we have previously reported. Tumor biopsies were taken within 1 month prior to infusion of CAR T cells. RNA expression was measured by RNAseq and/or a Nanostring IO360 panel consisting of 770 genes found in the tumor microenvironment (TME) in cancer. Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Mouse CAR T cell studies utilized mouse CD19-targeted CAR T cells derived from C57BL/6 splenocytes and cultured in vitro with myeloid cells and target cells to evaluate cytotoxicity and/or cytokine secretion. Elicited mouse macrophages were collected from peritoneal fluid 4 days after IP injection of 3% Brewer's thioglycollate medium. In vivo studies with mouse CD19-targeted CAR T cells were performed in IL2Ra-/- mice given cyclophosphamide as a pre-conditioning chemotherapy followed by adoptive transfer and analyses for CAR T cell and B cell persistence, as well as cytokines. Results: Of 58 patients undergoing CD19 CAR T cell therapy for LBCL, 8 (14%) had severe (grade 3 or higher) CRS and 16 (28%) had severe (grade 3 or higher) neurotoxicity. At baseline, peripheral blood levels of IL-6, IFN-γ, IL-15 and ferritin were significantly higher in patients who would subsequently experience severe CRS and severe neurotoxicity. Confirming our recent animal model of CRS we determined that peak serum catecholamine levels were higher in patients experiencing severe CRS. To identify if myeloid cells potentiate cytokine release we co-cultured CAR T cells with CD19 target and macrophages obtained from elicited mouse peritoneum. When these macrophages were added, IL-6 release from CAR T cells significantly increased compared to when macrophages were absent. Next, we studied the baseline TME in LBCL CAR T patients. Of 36 patients, 10 (27%) experienced severe neurotoxicity following CAR T cell therapy. By cell type score, the severe neurotoxicity group had a lower expression of genes associated with T cells overall and specifically Tregs. Also significantly lower in the severe neurotoxicity group were T cell genes including multiple subunits of CD3, CD3ζ, FOXP3, ICOS, CD62L and others. Association of increased T cell infiltration in the TME with low neurotoxicity raised the possibility that suppressive T cell subsets play a role in limiting toxicity post-CAR T cell therapy. To test this hypothesis, we injected CD19-targeted CAR T cells into an immune competent mouse model of Treg depletion (IL2Ra-/-) with established CD19+ leukemia. Treg deficient mice experienced a massive cytokine release after CAR T infusion and died prematurely due to CAR T toxicity compared to control mice with Tregs intact. Conclusions: Our observations suggest that the incidence of severe toxicity following CD19 CAR T cell therapy is influenced by baseline characteristics that are present prior to the infusion of CAR T cells. These include systemic inflammation characterized by high cytokine levels and a TME notable for a lack of infiltrating T cells. We posit a model whereby inflammation primes myeloid cells that are further activated upon CAR T cell infusion to release toxic amounts of cytokines and catecholamines. T cell subsets in the TME may modulate CAR T cells at the site of antigen encounter and prevent excessive CAR T activation. Reducing systemic inflammation or encouraging T cell infiltration into tumor prior to CAR T infusion are potential strategies for lowering the toxicity associated with CAR T therapy. Disclosures Jain: Kite/Gilead: Consultancy. Chavez:Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Novartis: Honoraria; Spectrum/Astrotech: Honoraria; Celgene/Juno: Honoraria; Kite/Gilead: Honoraria; Incyte: Research Funding; Jazz Pharmaceuticals: Research Funding; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; AstraZeneca: Honoraria. Bachmeier:Kite/Gilead: Speakers Bureau. Mullinax:Iovance: Research Funding. Locke:Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy; Kite: Other: Scientific Advisor. Davila:Anixa: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Celgene: Research Funding; Atara: Research Funding; Bellicum: Consultancy.

Description: Mutational analysis has established that the cytoplasmic tail of the integrin β3 subunit binds c-Src (termed as Src in this study) and is critical for bidirectional integrin signaling. Here we show in washed human platelets that a cell-permeable, myristoylated RGT peptide (myr-RGT) corresponding to the integrin β3 C-terminal sequence dose-dependently inhibited stable platelet adhesion and spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent second wave of platelet aggregation induced by adenosine diphosphate, ristocetin, or thrombin. Thus, myr-RGT inhibited integrin outside-in signaling. In contrast, myr-RGT had no inhibitory effect on adenosine diphosphate-induced soluble fibrinogen binding to platelets that is dependent on integrin inside-out signaling. Furthermore, the RGT peptide induced dissociation of Src from integrin β3 and dose-dependently inhibited the purified recombinant β3 cytoplasmic domain binding to Src-SH3. In addition, phosphorylation of the β3 cytoplasmic tyrosines, Y747 and Y759, was inhibited by myr-RGT. These data indicate an important role for β3-Src interaction in outside-in signaling. Thus, in intact human platelets, disruption of the association of Src with β3 and selective blockade of integrin αIIbβ3 outside-in signaling by myr-RGT suggest a potential new antithrombotic strategy.

Description: Background: c-MYC is a transcription factor that promotes oncogenesis by activating and repressing its target genes that control cell growth, metabolism, and proliferation. MYC is deregulated in a large proportion of aggressive B-cell lymphomas. A typical example is the Double-Hit Lymphoma (DHL) and Double-Expression Lymphoma (DEL) which present with a rapidly progressing clinical course, refractory to treatment, poor clinical outcome, and currently considered incurable. Nevertheless, MYC is considered as an "undruggable" target since it has no "active site" amenable to binding by conventional small molecule inhibitors. Moreover, MYC has a broad spectrum of functions in cell proliferation, survival, metabolism, and others, so direct inhibition would likely cause severe side effects. Besides direct inhibition, another practical strategy is to target druggable proteins that are essential for the viability of MYC-driven tumors, inducing MYC-dependent "synthetic lethality". The advantage of such approach is a capability of killing tumor cells discriminately, while leaving non-tumor cells intact or less influenced. This study is designed to identify such targets and explore practical novel strategies to treat MYC-driven lymphomas, especially DHL/DEL. Methods and Results: By integrating activity-based proteomic profiling and drug screens in isogenic MYC on/off lymphoma cells, we identified polo-like kinase-1 (PLK1) as an essential regulator of the MYC-dependent kinome in DHL/DEL. Notably, PLK1 was expressed at high levels in DHL, correlated with MYC expression and connoted poor outcome. Further, PLK1 is directly activated by MYC on transcriptional level and in turn, PLK1 signaling augmented MYC protein stability by promoting its phosphorylation and suppressing its degradation. Thus, MYC and PLK1 form a feed-forward circuit in lymphoma cells. Finally, both in vitro and in vivo studies demonstrated that inhibition of PLK1 triggered degradation of MYC and of the anti-apoptotic protein MCL1, and PLK1 inhibitors showed synergy with BCL-2 antagonists in blocking DHL/DEL cell growth, survival, and tumorigenicity. These data support that PLK1 is a promising therapeutic target in MYC-driven lymphomas. Brief summary: Functional pharmacoproteomics identified PLK1 as a therapeutic vulnerability for MYC-driven lymphoma, which was a synthetic lethal for DHL/DEL when targeted with BCL-2 inhibitors. Disclosures Vose: Roche: Honoraria; Merck Sharp & Dohme Corp.: Research Funding; Acerta Pharma: Research Funding; Seattle Genetics, Inc.: Research Funding; Novartis: Honoraria, Research Funding; Kite Pharma: Research Funding; Bristol Myers Squibb: Research Funding; Epizyme: Honoraria; Legend Pharmaceuticals: Honoraria; Abbvie: Honoraria; Celgene: Research Funding; Incyte Corp.: Research Funding.

Description: Abstract 4710 Lentiviral vectors (LVs) directly delivered to the bone marrow by intra-bone marrow (iBM) injection can efficiently transduce hematopoietic stem cells (HSCs) for treating genetic blood disorders. Our previous study showed that FVIII activity in the plasma of hemophilia A (HemA) mice achieved nearly normal levels one week after being treated with iBM injection of LVs containing a B-domain variant human factor VIII (hFVIII/N6) gene driven by a human elongation factor-1 promoter (E-LV). However, plasma FVIII activity fell very quickly with concomitant generation of anti-FVIII antibodies. In this study, to prevent anti-FVIII immune responses in the treated HemA mice, we treated three groups of mice with intraperitoneal injection of 200 μL of immunomodulatory protocols including IL-2/IL-2 mAb complexes (IL-2 Complex, 1 μg IL-2 mixed with 5 μg anti-IL-2 mAb per mouse on day −5, −4, and −3), anti-mouse CD3ε mAb (20 μg per mouse on day −2, −1, 0, 1 and 2), and rabbit anti-mouse thymocyte IgG (ATG, 0.6 mg per mouse on day −1 and 0), respectively. The mice were treated with 5 μL E-LV (7 × 107 TU/mL) on day 0. The peak plasma FVIII levels in 0/4 E-LV only treated mice, 3/5 IL-2 Complex pretreated mice, 2/3 anti-CD3ε pretreated mice and 4/5 ATS pretreated mice showed up one month after treatment. 2/4 E-LV only treated mice, 2/5 IL-2 Complex pretreated mice, 1/3 anti-CD3ε pretreated mice and 3/5 ATS pretreated mice generated anti-FVIII antibodies two months after treatment. These data indicated that all three immunomodulatory protocols protected E-LV transduced cells from eradication mediated by cytotoxic T cells in the early stage after treatment; however these protocols did not prevent the production of anti-FVIII antibodies at later time points. Next, we employed 10 HemA/Foxp3-Tg mice in which all T cells over-expressed Foxp3 as an immunosuppressive mouse model and treated them with low (5 μL) or high (25 μL) dosage of E-LVs, respectively. None of the 10 treated HemA/Foxp3-Tg produced anti-FVIII antibodies however plasma FVIII levels in all 10 mice fell to low levels one month after treatment. Thus, increase of regulatory T cells could not totally inhibit cytotoxic T cells to lyse transduced cells, whereas they prevented anti-FVIII antibody production. In order to examine whether LV-transduced cells were destroyed in the early stage post-injection, we treated HemA mice with 3 μL of LVs (4.6 × 1010 TU/mL) containing GFP gene driven by a modified myeloid proliferative sarcoma virus (MND) promoter and tracked GFP expression in bone marrow cells overtime. The percentage of GFP positive cells reached highest on day 15 post-injection, and then fell quickly afterwards, while GFP positive cells (0.04% of total bone marrow cells) were still detectable on day 94. Therefore, in order to achieve persistent transgene expression of hFVIII in the HemA mice treated with intra-bone marrow injection of E-LVs, cytotoxic-T-cell-mediated elimination of the transduced cells needs to be inhibited in the early stage and formation of anti-FVIII antibody can be eliminated by increasing FVIII-specific Treg cell populations. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Platelets may comprise an ideal vehicle for delivering factor VIII (FVIII) in hemophilia A (HemA) as FVIII stored in platelet α-granules is protected from neutralization by inhibitory antibodies and, during bleeding, activated platelets locally excrete their contents to promote clot formation. In our previous study, it was demonstrated that intraosseous (IO) infusion of lentiviral vectors (LVs) carrying a transgene encoding human factor VIII variant (BDDhFVIII/N6; abbreviated as F8) driven by a megakaryocyte-specific promoter (Gp1bα) successfully transduced hematopoietic stem cells (HSCs) in HemA mice without the requirement of preconditioning as in ex vivo gene therapy. FVIII expressed and then stored in platelets can partially correct the HemA phenotype over 5 months in mice with or without pre-existing inhibitors. Methods: In this study, we aimed at enhancing transgene expression by two strategies. One was to improve the efficiency of human FVIII cDNA by testing a new human FVIII variant (a kind gift from Dr. Weidong Xiao), F8X10K12 (a 10-amino acid change in the A1 domain and a 12-amino acid change in the light chain). The other was to enhance LV transduction efficiency by suppressing the innate and adaptive immune responses against LVs and LV-transduced cells using pharmacological agents. Results: We first tested the FVIII expression levels from two human FVIII variants by hydrodynamic injection of plasmids driven by a human elongation factor-1 promoter (pEF1α-F8X10K12 or pEF1α-F8, 50 μg/mouse, n=8), respectively. Compared with F8, F8X10K12 produced a 25-fold increase (147±27% vs 3,734±477%) in the clotting activity determined by an aPTT assay on day 4 post injection. Based on this result, two LVs containing F8X10K12 or F8 transgene driven by EF1α promoter (E-F8X10K12-LV or E-F8-LV) were constructed and used to transduce 293T cells, respectively. Flow cytometry data showed that E-F8X10K12-LV produced a significant increase of hFVIII+ 293T cells (77.8% vs 15%) and MFI (795 vs 541) compared to E-F8-LV at the same doses (Figure 1). These results indicated that F8X10K12 may further enhance FVIII gene expression for more effective therapy. Two LVs containing F8X10K12 or F8 transgene driven by Gp1bα promoter (G-F8X10K12-LV or G-F8-LV) were subsequently generated. Secondly, the immune competent C57BL6 mice were pretreated with both dexamethasone (IP, 5 mg/kg at -24h, -4h, 4h and 24h) and anti-CD8α monoclonal antibody (mAb) (IP, 4 mg/kg on day -1, 4, 11, 16 and 21). IO infusion of GFP-LVs (1.1×108 i.f.u./mouse) driven by a ubiquitous MND promoter was performed on day 0 (Figure 2a). On day 7, drugs + LVs treated mice (n=3) produced higher numbers of GFP+ total bone marrow cells (11.8±2.1% vs 6.9±3.1%, P =0.005) and GFP+ Lineage- Sca1+ cKit+ HSCs (48.3±6.1% vs 44.4±17.2%, P =0.31) compared with LV-only treated mice (n=3) (Figure 2b). Most importantly, in the long term, higher numbers of GFP+ cells (2.4±0.4% vs 0.5±0.1%, P

Description: Combined factor V and factor VIII deficiency is a rare disorder associated with relatively mild bleeding diathesis. Shao and colleagues elucidate the double role of factor V as both a pro- and anticoagulant protein, demonstrating that decreased factor V may ameliorate factor VIII deficiency through decreasing the level of tissue factor pathway inhibitor.