scholarly journals GLT1 gene delivery based on bone marrow-derived cells ameliorates motor function and survival in a mouse model of ALS

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Natsuko Ohashi ◽  
Tomoya Terashima ◽  
Miwako Katagi ◽  
Yuki Nakae ◽  
Junko Okano ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Mouse Model ◽  
Lentiviral Vector ◽  
White Blood Cells ◽  
Glutamate Toxicity ◽  
Pathological Lesion ◽  
Arginase 1 ◽  
Therapy Strategy

AbstractAmyotrophic lateral sclerosis (ALS) is an intractable neurodegenerative disease. CD68-positive bone marrow (BM)-derived cells (BMDCs) accumulate in the pathological lesion in the SOD1(G93A) ALS mouse model after BM transplantation (BMT). Therefore, we investigated whether BMDCs can be applied as gene carriers for cell-based gene therapy by employing the accumulation of BMDCs. In ALS mice, YFP reporter signals were observed in 12–14% of white blood cells (WBCs) and in the spinal cord via transplantation of BM after lentiviral vector (LV) infection. After confirmation of gene transduction by LV with the CD68 promoter in 4–7% of WBCs and in the spinal cord of ALS mice, BM cells were infected with LVs expressing glutamate transporter (GLT) 1 that protects neurons from glutamate toxicity, driven by the CD68 promoter, which were transplanted into ALS mice. The treated mice showed improvement of motor behaviors and prolonged survival. Additionally, interleukin (IL)-1β was significantly suppressed, and IL-4, arginase 1, and FIZZ were significantly increased in the mice. These results suggested that GLT1 expression by BMDCs improved the spinal cord environment. Therefore, our gene therapy strategy may be applied to treat neurodegenerative diseases such as ALS in which BMDCs accumulate in the pathological lesion by BMT.

scholarly journals Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector

Haematologica ◽  
2021 ◽  
Author(s):  
Yang Liu ◽  
Maria Dahl ◽  
Shubhranshu Debnath ◽  
Michael Rothe ◽  
Emma M. Smith ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Mouse Model ◽  
Cord Blood ◽  
Lentiviral Vector ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Clinical Gene Therapy ◽  
Cord Blood Cells

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder with pure red blood cell aplasia associated with physical malformations and a predisposition to cancer. Twenty-five percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1α short promoter, that can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19-deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice longterm and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.

scholarly journals Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Tugba Mehmetoglu-Gurbuz ◽  
Rose Yeh ◽  
Himanshu Garg ◽  
Anjali Joshi
Keyword(s):  
Gene Therapy ◽  
Lentiviral Vector ◽  
Suicide Gene ◽  
Vector System ◽  
Tat Protein ◽  
Ccr5 Gene ◽  
Hematopoietic Stem ◽  
Therapy Strategy ◽  
Hiv Tat ◽  
A Cell

Abstract Background Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone. Methods A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein. Results Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir. Conclusions Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.

scholarly journals A self-inactivating lentiviral vector for SCID-X1 gene therapy that does not activate LMO2 expression in human T cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 900-908 ◽  
Author(s):  
Sheng Zhou ◽  
Disha Mody ◽  
Suk See DeRavin ◽  
Julia Hauer ◽  
Taihe Lu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Protein Expression ◽  
Genomic Sequence ◽  
Bone Marrow Cells ◽  
Lentiviral Vector ◽  
Severe Combined Immunodeficiency ◽  
Vitro Assay ◽  
Activation Assay ◽  
Marrow Cells

Abstract To develop safer and more effective vectors for gene therapy of X-linked severe combined immunodeficiency (SCID-X1), we have evaluated new self-inactivating lentiviral vectors based on the HIV virus. The CL20i4-hγc-Revgen vector contains the entire human common γ chain (γc) genomic sequence driven by the γc promoter. The CL20i4-EF1α-hγcOPT vector uses a promoter fragment from the eukaryotic elongation factor alpha (EF1α) gene to express a codon-optimized human γc cDNA. Both vectors contain a 400-bp insulator fragment from the chicken β-globin locus within the self-inactivating long-terminal repeat. Transduction of bone marrow cells using either of these vectors restored T, B, and natural killer lymphocyte development and function in a mouse SCID-X1 transplantation model. Transduction of human CD34+ bone marrow cells from SCID-X1 patients with either vector restored T-cell development in an in vitro assay. In safety studies using a Jurkat LMO2 activation assay, only the CL20i4-EF1α-hγcOPT vector lacked the ability to transactivate LMO2 protein expression, whereas the CL20i4-hγc-Revgen vector significantly activated LMO2 protein expression. In addition, the CL20i4-EF1α-hγcOPT vector has not caused any tumors in transplanted mice. We conclude that the CL20i4-EF1α-hγcOPT vector may be suitable for testing in a clinical trial based on these preclinical demonstrations of efficacy and safety.

scholarly journals 358. Lentiviral Vector Integration in Mouse Bone Marrow Stem Cells Undergoing Gene Therapy

2006 ◽  
Vol 13 ◽  
pp. S136 ◽  
Author(s):  
Xiaolin Wu ◽  
Weijing He ◽  
Shawn M. Burgess ◽  
Robert A. Clark ◽  
Senlin Li
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Lentiviral Vector ◽  
Bone Marrow Stem Cells ◽  
Mouse Bone Marrow ◽  
Vector Integration

Gene Therapy Corrects the Lethal Bone Marrow Failure in a Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 513-513
Author(s):  
Pekka Jaako ◽  
Shubhranshu Debnath ◽  
Karin Olsson ◽  
Axel Schambach ◽  
Christopher Baum ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Stem And Progenitor Cells

Abstract Abstract 513 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical abnormalities and predisposition to cancer. Mutations in genes that encode ribosomal proteins have been identified in approximately 60–70 % of the patients. Among these genes, ribosomal protein S19 (RPS19) is the most common DBA gene (25 % of the cases). Current DBA therapies involve risks for serious side effects and a high proportion of deaths are treatment-related underscoring the need for novel therapies. We have previously demonstrated that enforced expression of RPS19 improves the proliferation, erythroid colony-forming potential and differentiation of patient derived RPS19-deficient hematopoietic progenitor cells in vitro (Hamaguchi, Blood 2002; Hamaguchi, Mol Ther 2003). Furthermore, RPS19 overexpression enhances the engraftment and erythroid differentiation of patient-derived hematopoietic stem and progenitor cells when transplanted into immunocompromised mice (Flygare, Exp Hematol 2008). Collectively these studies suggest the feasibility of gene therapy in the treatment of RPS19-deficient DBA. In the current project we have assessed the therapeutic efficacy of gene therapy using a mouse model for RPS19-deficient DBA (Jaako, Blood 2011; Jaako, Blood 2012). This model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of Collagen A1 gene. Transgenic animals were bred either heterozygous or homozygous for the shRNA-D in order to generate two models with intermediate or severe Rps19 deficiency, respectively. Indeed, following transplantation, the administration of doxycycline to the recipients with homozygous shRNA-D bone marrow results in an acute and lethal bone marrow failure, while the heterozygous shRNA-D recipients develop a mild and chronic phenotype. We employed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the SFFV promoter, followed by IRES and GFP (SFFV-RPS19). A similar vector without the RPS19 cDNA was used as a control (SFFV-GFP). To assess the therapeutic potential of the SFFV-RPS19 vector in vivo, transduced c-Kit enriched bone marrow cells from control and homozygous shRNA-D mice were injected into lethally irradiated wild-type mice. Based on the percentage of GFP-positive cells, transduction efficiencies varied between 40 % and 60 %. Three months after transplantation, recipient mice were administered doxycycline in order to induce Rps19 deficiency. After two weeks of doxycycline administration, the recipients transplanted with SFFV-RPS19 or SFFV-GFP control cells showed no differences in blood cellularity. Remarkably, at the same time-point the recipients with SFFV-GFP homozygous shRNA-D bone marrow showed a dramatic decrease in blood cellularity that led to death, while the recipients with SFFV-RPS19 shRNA-D bone marrow showed nearly normal blood cellularity. These results demonstrate the potential of enforced expression of RPS19 to reverse the severe anemia and bone marrow failure in DBA. To assess the reconstitution advantage of transduced hematopoietic stem and progenitor cells with time, we performed similar experiments with heterozygous shRNA-D bone marrow cells. We monitored the percentage of GFP-positive myeloid cells in the peripheral blood, which provides a dynamic read-out for bone marrow activity. After four months of doxycycline administration, the mean percentage of GFP-positive cells in the recipients with SFFV-RPS19 heterozygous shRNA-D bone marrow increased to 97 %, while no similar advantage was observed in the recipients with SFFV-RPS19 or SFFV-GFP control bone marrow, or SFFV-GFP heterozygous shRNA-D bone marrow. Consistently, SFFV-RPS19 conferred a reconstitution advantage over the non-transduced cells in the bone marrow. Furthermore, SFFV-RPS19 reversed the hypocellular bone marrow observed in the SFFV-GFP heterozygous shRNA-D recipients. Taken together, using mouse models for RPS19-deficient DBA, we demonstrate that the enforced expression of RPS19 rescues the lethal bone marrow failure and confers a strong reconstitution advantage in vivo. These results provide a proof-of-principle for gene therapy in the treatment of RPS19-deficient DBA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Leukemia Risk Gene ARID5B is a Crucial Regulator of B-Cell Development

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 385-385 ◽  
Author(s):  
Charnise Goodings-Harris ◽  
Hui Zhang ◽  
Xuijie Zhao ◽  
Heng Xu ◽  
Omar I Abdel-Wahab ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Sudden Death ◽  
Mouse Model ◽  
B Cell ◽  
Blood Cells ◽  
Association Studies ◽  
White Blood Cells ◽  
Cell Populations ◽  
Spleen Volume ◽  
Marrow Cellularity

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Genome wide association studies by us and others have identified germline genetic variants at the ARID5B locus that strongly influence susceptibility to ALL (Nat Genet 2009, J Clin Oncol 2012). Despite compelling results from these genetic association studies, the molecular mechanisms by which ARID5B contributes to ALL pathogenesis remains largely unknown. ARID5B is a member of the ARID transcription family characterized by a conservative AT rich binding domain, but the physiological functions of ARID5B are poorly understood. Therefore we sought to develop mouse models to comprehensively characterize the roles of ARID5B in normal and malignant hematopoiesis. To this end, we first established a Vav-1 specific Arid5b overexpression (AOE) mouse model. Mice were designed with a tetO element knocked in before the start codon of Arid5b to create an Arid5b tetO mouse. Arid5b tetO mice were then crossed with Vav-1-tTA mice and, overexpression of Arid5b confirmed in the hematopoietic system of progenies with the desired genotype. At 6-8 weeks old complete blood counts (CBC) analysis revealed that AOE mice had a large decrease in white blood cells (WBCs) and a slight reduction in red blood cells (RBCs) and hemoglobin (Hb). We also noted a significant reduction in the bone marrow cellularity of our Arid5b overexpressing mice. To further characterize our AOE mouse model we used flow cytometry to quantify the proportions of mature as well as various stem and progenitor cell populations during hematopoiesis. We found that AOE mice showed an increase in the MegE biased MPP2 (LSK flt3−/CD150+/CD48+) population and a decrease in the lymphoid biased MPP4 (LSK (Flt3+/CD150−/CD48+/−) population. Overexpression of Arid5b resulted in a significant reduction in the proportion of all bone marrow B cell populations (Hardy Fraction A-F) (Figure 1A) and B220+ cells in the spleen. In vitro methylcellulose colony forming assays further revealed a loss of functional pre B lymphoid progenitor in AOE bone marrow. To determine if the hematopoietic defects seen in younger AOE mice was due to a delay in hematopoiesis we aged AOE mice. We found that as we aged AOE mice there was still a reduction in bone marrow cellularity and all bone marrow B cell populations. Surprisingly, as mice were aged we found that AOE mice were prone to sudden death and displayed a dramatic reduction in overall survival when compared to wildtype littermates with a mean survival age of 8 months (Figure 1B). At the time of death, we found that AOE mice presented with severely enlarged spleens. To predict the death of AOE mice we performed ultrasounds to track spleen volume. Based on ultrasounds performed on AOE mice and wildtype littermates, AOE mice spleens become larger than those of their littermates as early as 6 months old, however spleen volume did not predict sudden death. In aged AOE mice, while the spleens were grossly enlarged and contained larger amounts of red pulp and increased Ter119+ cells, there was still a reduction in the proportion of B cells and no increase in the proportions of other white blood cells (Figure 1D). Older AOE mice exhibited anemia, hypergammaglobulinemia, and splenomegaly. By 30 weeks, defects in B cell proportions were seen in the lymph nodes of AOE mice via immunohistochemistry (Figure 1C). Compared to wildtype littermates, AOE mice displayed reduced white blood cells, red blood cells, and platelets (Figure 1D). The anemia was associated with higher reticulocyte numbers and increased serum erythropoietin concentration. The life span of erythrocytes from AOE mice was less than that of wildtype littermates. Together, these results indicated that Arid5b plays an important role in B lymphopoiesis and erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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