This was a single-arm, multicenter, open-label phase I trial. Lentiviral vectors (LV) carrying the ABCD1 gene (LV-ABCD1) was directly injected into the brain of patients with childhood cerebral adrenoleukodystrophy (CCALD), and multi-site injection was performed. The injection dose increased from 200 to 1600 μL (vector titer: 1×109 transduction units per mL (TU/mL)), and the average dose per kilogram body weight ranges from 8 to 63.6 μL/kg. The primary endpoint was safety, dose-exploration and immunogenicity and the secondary endpoint was initial evaluation of efficacy and the expression of ABCD1 protein. A total of 7 patients participated in this phase I study and were followed for 1 year. No injection-related serious adverse event or death occurred. Common adverse events associated with the injection were irritability (71%, 5/7) and fever (37.2-38.5 ℃, 57%, 4/7). Adverse events were mild and self-limited, or resolved within 3 d of symptomatic treatment. The maximal tolerable dose is 1600 μL. In 5 cases (83.3%, 5/6), no lentivirus associated antibodies were detected. The overall survival at 1-year was 100%. The ABCD1 protein expression was detected in neutrophils, monocytes and lymphocytes. This study suggests that the intracerebral injection of LV-ABCD1 for CCALD is safe and can achieve successful LV transduction in vivo; even the maximal dose did not increase the risk of adverse events. Furthermore, the direct LV-ABCD1 injection displayed low immunogenicity. In addition, the effectiveness of intracerebral LV-ABCD1 injection has been preliminarily demonstrated while further investigation is needed. This study has been registered in the Chinese Clinical Trial Registry (https://www.chictr.org.cn/, registration number: ChiCTR1900026649).

PDF file - 66K, Validation of Wnt/beta-catenin reporter in CT-1 pCCSCs

Background: Chimeric antigen receptor (CAR) T cells therapy is highly effective in the treatment of B-cell lymphoma, providing alternative therapeutic options for patients who failed to respond to conventional treatment or relapse. Despite impressive progress, more than 30-50% of patients treated with CD19 CAR-T cells experienced progressive disease. Antigen escape is one of the common causes of relapse, especially CD19-negative relapse. Absent or decreased cell surface CD19 as a mechanism of resistance after CD19 CAR-T cells treatment. CD70 is a novel and promising therapeutic target due to the restricted expression pattern in normal tissues and over expression in some lymphoma tissues. Our center explored the efficacy and safety of CAR-T cells therapy with dual targeting of CD19 and CD70 in the treatment of r/r lymphoma. Patients and Methods: The study included 8 relapsed/refractory diffuse large B-cell lymphoma patients, all patients have exhausted all available treatment options with progressive or stable disease and life expectancy >2 months were enrolled in the study. All patients expressed strong positivity of CD19 and CD70, all of patient details can be found in the table (Fig.1). Autologous T cells were apheresis collected and transduced with an apoptosis-inducible, safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/CD27/CD3z-iCasp9. All patients received cyclophosphamide/fludarabine chemotherapy conditioning 1-2 days before infusions of CAR-T cells. The quality of apheresis cells, efficiencies of gene transfer and T cell proliferation, CAR-T infusion dose and blood CAR copies were quantitatively documented. Results: In the first months after CAR-T cells infusion, 75.0% (6/8) of patients achieved complete response (CR), while 12.5% (1/8) of patients evaluated as partial response (PR) and 12.5% (1/8) of patients as progressive disease (PD), overall response rate (ORR) is 87.5% (7/8). In the evaluation of safety, 37.5% (3/8) of patients experienced cytokine release syndrome (CRS), and 25% (2/8) of patients experienced grade 1 CRS and 12.5% (1/8) of patients experienced grade 2 CRS, no case of severe CRS defined as≥grade 3 and no immune effector cell-associated neurotoxicity syndrome (ICANS) occurred. After a median follow-up of 19.9 months, 50% (4/8) of patients maintained CR, while 37.5% (3/8) of patients were relapsed, with a duration of response (DOR) of 50% (4/8), median DFS was 10.5 months and median OS was not reached (Fig.2). Conclusions: In summary, our research data confirms that the efficacy and safety of CD19/CD70 dual targeted CAR-T cells infusion, continued follow-up will determine whether the CD19/CD70 CAR-T cells therapy can obtain long term overall survival in our study. How to further improve the efficacy and safety of dual target CAR-T is still worth exploring. It's needed to optimize multi-specific targeting by CAR-T cells to improve the efficacy both in B cell malignancies and other hematological malignancies.

PDF file - 120K, Supplementary methods include information on Wnt reporter and beta-Catenin shRNA knockdown constructs, real-time PCR, tumor dissociation, FACS, immunostaining and western blotting

PDF file - 1.2MB, FACS profile and experimental overview

PDF file - 995K, Comparison of primary versus secondary Wnt(high) CT-2 tumor xenografts

PDF file - 867K, Beta-catenin knockdown

PDF file - 383K, Proposed CCSC hierarchy in CAC

Chronic granulomatous disease (CGD) is a rare congenital immunodeficiency characterized by a defect in nicotinamide adenine dinucleotide phosphate oxidase required for phagocytosis. Hematopoietic stem cell (HSC) transplantation is currently the only curative treatment, but it is ladened with morbidities and mortality. Gene therapy is a promising treatment for CGD. However, if not properly designed, the gene therapy approach may not be successful. We engineered lentiviral vectors (LVs) carrying a universal promoter (EF1a) and two myeloid-specific promoters (miR223 and CD68) to drive the expression of green fluorescence protein (GFP) or CYBB, one of the key defective genes causing CGD. Tissue-specific LV expression was investigated in vitro and in a CGD mouse model. We compared GFP expression in both myeloid differentiated and undifferentiated HSCs. The CGD mice were transplanted with LV-modified mouse HSCs to investigate expression of CYBB and restoration of reactive oxygen species. The LV promoters were further compared under low and high-transgenic conditions to assess safety and therapeutic efficacy. A pneumonia disease model based on pathogenic Staphylococcus aureus challenge was established to assess the survival rate and body weight change. All three promoters demonstrated ectopic CYBB expression in vitro and in vivo. The EF1a promoter showed the highest expression of GFP or CYBB in transduced cells, including HSCs without cytotoxicity, whereas the LV-miR223 showed the highest transgene delivery efficiency with high myeloid specificity. Importantly, under low-transgenic condition, only the LV-EF1a-CYBB showed high antibacterial activity in vivo.

Abstract Chronic granulomatous disease (CGD) is a congenital immunodeficiency characterized by lack of reactive oxygen species in phagocytes. We developed an in vivo gene therapy strategy based on intravenous (iv) injection of lentiviral vectors (LVs) in X-CGD mice. A non-myeloablative chemo-conditioning regimen using busulfan, cyclophosphamide and dexamethasone was developed to improve iv LV gene delivery efficiency. The X-CGD mice received two LVs injections. After the second injection, antibody response to LV particle-associated p24-protein was examined by Western blot. We detected increased gene transfer without anti-p24 antibody response. However, the blood vector copy number (VCN) was gradually reduced after 3–12 months. To improve gene delivery into hematopoietic stem cells (HSCs), the mice were treated with AMD3100 to mobilize HSCs before LV injection. To confirm HSCs gene transfer, we transplanted the HSCs from the LV-CYBB-treated CGD mice into untreated CGD mice. The result showed successful passage of LV-CYBB HSCs to recipient mice. Thus, by combining chemo-conditioning and AMD3100 mobilization prior to the iv LV injection, improved in vivo long-term LV gene transfer into HSCs could be established. This improved iv LV gene delivery strategy could reduce both the risk and the cost of CGD gene therapy with great potential in translational applications.

Background: Chimeric antigen receptor T-cells (CAR-T) targeting CD19 therapy is reported to induce 83-93% response in relapsed/refractory acute lymphoblastic leukemia (r/r-ALL) in adults. However, 30-60% of patients relapse after CAR-T treatment, of which 14-25% CD19-negative relapse. In spite of CAR-T cells persistence, CD19 absent causing tumors that evade CAR-T cells mediated recognition and clearance. For CD19-negative relapse, it is necessary to explore more effective targets. There are some research proved that compared with single-targeted CARs, CD19/CD22 dual-targeted CARs induce more IFN-γ and IL-2 in vitro and eradicate patient-derived xenografts (PDX) produced with CD19-negative relapse of CD19-directing CAR-T treatment. Therefore, our study explored the efficacy and safety of clinical studies on mixture of CD19 CAR-T and CD22 CAR-T cells in the treatment of r/r ALL in adults. Patients and Methods: CD3 + cells were selected from the apheresis PBMC and activated before lentiviral CAR T cells infection. The cells were transduced with a caspase9-inducible, safety-engineered lentivector CAR containing anti-CD19 or -CD22 scFv fused with multiple intracellular signaling domains: CD28/CD27/CD3z-iCasp9. The quality of apheresis cells, gene transfer and T cell proliferation efficiencies, and effective CAR-T infusion dose were quantitatively scored and documented. All enrolled patients expressed CD19 and CD22 positively. All patients received fludarabine (FLU) and cyclophosphamide (CTX) conditioning chemotherapy (FLU 30mg/m 2, d1-3; CTX 500mg/m 2, d1-3) before CAR-T infusions. Results: The study included 16 r/r ALL patients, median age is 26 years, and median of previous theraples is 6, all of patient details can be found in the table (Fig.1). In the first month after CD19/CD22 CAR-T cells infusion, total of 93.8% (15/16) of patients achieved complete response (CR), 1 case (6.25%) was evaluated as non-remission (NR). In the evaluation of safety, 68.75% (11/16) of patients did not experience cytokine release syndrome (CRS), 25% of patients (4/16) occurred grade 1 CRS and 6.25% (1/16) of patients occurred grade 2 CRS, no case of severe CRS defined as≥grade 3 and no any immune effector cell-associated neurotoxicity syndrome (ICANS) occurred. After a median follow-up of 9.9 months, 4 patients maintained CR, while other 11 patients were relapsed, with a duration of response (DOR) of 25% (4/16), median LFS was 6.1 months and median OS were not reached (Fig.2). Conclusions: In summary, CD19/CD22 exhibited a manageable safety but limited antileukemia activity. Our study showed that mixture of CD19 CAR-T and CD22 CAR-T cells infusion is fail to expected to compensate for the treatment of relapsed in r/r ALL and prolonging the leukemia-free survival of patients. The low-efficacy of dual-targeted CAR-T cells may be caused by the CAR19 and CAR22 cells interfere with each other and weaken the CAR-T amplification in vivo. From our data, it seems that the timing of treatment and tumor burden status may play a more important role rather than new targets CAR-T cells infusion for r/r ALL in adults.

PDF file - 91K, Characteristics of patients from whom colon tissues were derived for immunostaining of beta-catenin and ALDH

PDF file - 857K, Primary CT-2 ALDH(high)-Wnt(low) tumor xenografts creates a phenocopy of an ALDH(high)-Wnt(high) tumor xenografts

Background: While complete response rate of CD19 CAR-T in diffuse large B-cell lymphoma (DLBCL) reached 40-60%, about 30%-50% of patients still face relapse, strategies that can enhance the therapeutic effects of CAR-T cells infusion should be actively explored. Bruton's tyrosine kinase (BTK) is an essential component of multiple signaling pathways including B-cell receptor signaling that regulate B cell proliferation, survival, and functions. These receptor signaling has been implicated in the survival of malignant B-cells making it a promising therapeutic target for various B-cell malignancies and inflammatory diseases. Different types of BTKi regulate T cell and B cell function through different signal pathways. Research has proved that CD19 CAR-T Cells in combination with ibrutinib for the treatment of relapsed/refractory aggressive B-cell lymphomas shows a high response rate and low level of adverse events, while zanubrutinib has not been reported. Therefore, our center explored the efficacy and safety of CD19 CAR-T Cells in combination with zanubrutinib for the strategy of relapsed/refractory DLBCL. Patients and Methods: The study included 6 r/r DLBCL patients, 4 patients have previously received 3-line or above treatment, and 2 patients began CAR-T treatment after 1-line chemotherapy. All patients received fludarabine (FLU) and cyclophosphamide (CTX) conditioning chemotherapy (FLU 30mg/m 2. d1-3; CTX 500mg/m 2, d1-3) before CAR-T infusions. T cells were apheresis collected and transduced with a lentiviral CD19 scFv CAR fused with intracellular signaling domains: CD28/CD27/CD3z-iCasp9. CAR-T cells were infused at dose of 2.0x10 6 cells/kg. The quality of apheresis cells, gene transfer and T cell proliferation efficiencies, and effective CAR-T infusion dose were quantitatively scored and documented. All patients began to combine with zanubrutinib after one month of CAR-T cells infusion. Results: After one month of CAR-T cells infusion, total of 50% (3/6) of patients achieved complete response (CR), while 50%(3/6) of patients were evaluated as partial response (PR), with an overall response rate (ORR) of 100%. Within 6 months after combined with zanubrutinib, all patients of PR achieved CR. In the evaluation of safety, 83% (5/6) of patients occurred grade 2 cytokine release syndrome (CRS), and 33.3% (2/6) patient occurred grade 1 immune effector cell-associated neurotoxicity syndrome (ICANS), no case of severe CRS defined as≥grade 3 occurred, 66.7% (4/6) of patients occurred grade 2 hematotoxicity and 33.3% (2/6) of patients occurred grade 4 hematotoxicity. After a median follow-up of 19.5 months, 6 patients maintained CR, with an duration of response (DOR) of 100%, median DFS and OS were not reached. All patients can be detected CAR-T cells sustain in peripheral blood mononuclear cells continuously until last follow-up date(Fig.2). Continued follow-up will confirm whether the CAR-T cells therapy combined with zanubrutinib can obtain long term overall survival in these study. Conclusions: Our study proved that CD19 CAR-T Cells combined with zanubrutinib for the strategy of relapsed/refractory DLBCL and as maintenance treatment showed strong efficacy with acceptable toxicity, the current results already support that zanubrutinib in the treatment of r/r DLBCL is expected to reduce the risk of relapsed, thereby prolonging the disease-free survival of patients, without increasing the incidence of adverse effects. More follow-up and clinical trials are needed to clarify the efficacy of Chimeric Antigen Receptor T Cells Therapy combined with zanubrutinib in the treatment of r/r DLBCL.

IntroductionAllogeneic hematopoietic stem cell transplantation (allo-HCT) is a standard treatment for relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). However, about 30-40% of patients still relapse after HCT. Chimeric antigen receptor-modified T-cell (CAR-T) therapy has been proven effective in the treatment of relapsed or refractory B-ALL.Patients and methodsWe report a cohort of 30 B-ALL patients, who relapsed after HCT and were enrolled in the 4SCAR2.0 study, receiving CD19 CAR-Ts alone (20 patients), or two types of CAR-Ts targeting CD19, CD22, CD38 or CD123 antigens (10 patients), depending on the tumor antigen expression profile. These patients had extramedullary (EM) relapse or bone marrow (BM) relapse, or both. Based on the GVHD history, donor chimerism, and the available T-cell source, 25 patients received allogeneic donor CAR-Ts, and 5 patients received autologous CAR-T treatment. ResultsAll 20 patients receiving a single CD19 CAR-T infusion achieved a minimal residual disease (MRD) remission within 60 days. The remaining 10 patients, due to low CD19 antigen expression profile, received 2 CAR-T products given on the same day, and 9 of 10 achieved complete remission (CR) and one had disease progression within 60 days. After CAR-T infusion, no cytokine release syndrome (CRS) was observed in 14 patients, and 16 patients experienced grade 1 CRS, and there was no neurotoxicity. Seventeen of the 30 patients who achieved remission (57%) remained in continuous remission following CAR-T treatment with a median follow-up period of 2 years and a median duration of remission of 12 months (range: 2.8 months - 67 months). Twelve out of 29 patients (41%) who achieved remission, subsequently relapsed at a median of 6.3 months (range: 2.8 months - 22.3 months) after CAR-T treatment. In summary, 29 patients (97%) achieved MRD negative remission within 60 days of therapy with a single or double CAR-T infusion, and seven patients remained in durable remission (7/30, 23%) after more than 2 years of follow-up. DiscussionThe tumor antigen profile-guided precision 4SCAR2.0 regimen for the treatment of r/r B-ALL after allo-HCT was highly effective with low toxicity. This approach warrants extended follow-up and further studies. Clinical trial registrationClinicalTrials.gov, identifier NCT03125577.

Abstract Background Hemophilia A (HA) is an X-linked monogenic disorder caused by deficiency of the factor VIII (FVIII) gene in the intrinsic coagulation cascade. The current protein replacement therapy (PRT) of HA has many limitations including short term effectiveness, high cost, and life-time treatment requirement. Gene therapy has become a promising treatment for HA. Orthotopic functional FVIII biosynthesis is critical to its coagulation activities. Methods To investigate targeted FVIII expression, we developed a series of advanced lentiviral vectors (LVs) carrying either a universal promoter (EF1α) or a variety of tissue-specific promoters, including endothelial-specific (VEC), endothelial and epithelial-specific (KDR), and megakaryocyte-specific (Gp and ITGA) promoters. Results To examine tissue specificity, the expression of a B-domain deleted human F8 (F8BDD) gene was tested in human endothelial and megakaryocytic cell lines. Functional assays demonstrated FVIII activities of LV-VEC-F8BDD and LV-ITGA-F8BDD in the therapeutic range in transduced endothelial and megakaryocytic cells, respectively. In F8 knockout mice (F8 KO mice, F8null mice), intravenous (iv) injection of LVs illustrated different degrees of phenotypic correction as well as anti-FVIII immune response for the different vectors. The iv delivery of LV-VEC-F8BDD and LV-Gp-F8BDD achieved 80% and 15% therapeutic FVIII activities over 180 days, respectively. Different from the other LV constructs, the LV-VEC-F8BDD displayed a low FVIII inhibitory response in the treated F8null mice. Conclusions The LV-VEC-F8BDD exhibited high LV packaging and delivery efficiencies, with endothelial specificity and low immunogenicity in the F8null mice, thus has a great potential for clinical applications.

PDF file - 2MB, Wnt activity in ALDH(high)-Wnt(high) and ALDH(high)-Wnt(low) primary tumor xenografts (1o)

PDF file - 3.7MB, Levels of Wnt/beta-catenin signaling in normal, colitis and CRC colons