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1.
Heliyon ; 9(9): e19435, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37810095

RESUMO

Selective T-cell depletion prior to cell or organ transplantation is considered a preconditioning regimen to induce tolerance and immunosuppression. An immunotoxin consisting of a recombinant anti-CD3 antibody conjugated with diphtheria toxin was used to eliminate T-cells. It showed significant T-cell depletion activity in the peripheral blood and lymph nodes in animal models used in previous studies. To date, a comprehensive evaluation of T-cell depletion and CD3 proliferation for all lymphoid tissues has not been conducted. Here, two rhesus macaques were administered A-dmDT390-SCFBdb (CD3-IT) intravenously at 25 µg/kg twice daily for four days. Samples were collected one day prior to and four days post administration. Flow cytometry and immunofluorescence staining were used to evaluate treatment efficiency accurately. Our preliminary results suggest that CD3-IT treatment may induce higher depletion of CD3 and CD4 T-cells in the lymph nodes and spleen, but is ineffective in the colon and thymus. The data showed a better elimination tendency of CD4 T-cells in the B-cell zone relative to the germinal center in the lymph nodes. Further, CD3-IT treatment may lead to a reduction in germinal center T follicular helper CD4 cells in the lymph nodes compared to healthy controls. The number of proliferating CD3 T-cell indicated that repopulation in different lymphoid tissues may occur four days post treatment. Our results provide insights into the differential efficacy of CD3-IT treatment and T-cell proliferation post treatment in different lymphoid tissues. Overall, CD3-IT treatment shows potential efficacy in depleting T-cells in the periphery, lymph nodes, and spleen, making it a viable preconditioning regimen for cell or organ transplantation. Our pilot study provides critical descriptive statistics and can contribute to the design of larger future studies.

2.
Mol Ther Methods Clin Dev ; 29: 483-493, 2023 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-37273902

RESUMO

CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system. Here, we investigated existence of immunity to SpCas9 and SaCas9 in control rhesus macaques (Macaca mulatta) alongside monkeys transplanted with either lentiviral transduced or CRISPR-SpCas9 ribonucleoprotein (RNP)-edited cells. We observed significant levels of Cas9 antibodies in the peripheral blood of all transplanted and non-transplanted control animals. Transplantation of ex vivo transduced or SpCas9-mediated BCL11A enhancer-edited cells did not alter the levels of Cas9 antibodies in rhesus monkeys. Following stimulation of peripheral blood cells with SpCas9 or SaCas9, neither Cas9-specific T cells nor cytokine induction were detected. Robust and durable editing frequencies and expression of high levels of fetal hemoglobin in BCL11A enhancer-edited rhesus monkeys with no evidence of an immune response (>3 years) provide an optimistic outlook for the use of ex vivo CRISPR-SpCas9 (RNP)-edited cells.

3.
Mol Ther Nucleic Acids ; 31: 452-465, 2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36852088

RESUMO

Transcriptional enhancers can be in physical proximity of their target genes via chromatin looping. The enhancer at the ß-globin locus (locus control region [LCR]) contacts the fetal-type (HBG) and adult-type (HBB) ß-globin genes during corresponding developmental stages. We have demonstrated previously that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the ß-globin locus as a model, we provide proof of concept at the organismal level that forced enhancer rewiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPCs) from mice bearing human ß-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus macaque erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene-regulatory elements may be used to alter gene expression to treat disease.

4.
Cell Rep Med ; 2(4): 100247, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33948577

RESUMO

Sickle cell disease (SCD) is caused by a 20A > T mutation in the ß-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34+ cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact ß-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34+ cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus ß-to-ßs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34+ cells 10-12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34+ HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.


Assuntos
Anemia Falciforme/genética , Antígenos CD34/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Xenoenxertos/imunologia , Macaca mulatta/genética , Animais , Edição de Genes/métodos , Marcação de Genes/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Hemoglobina Falciforme/genética , Humanos , Camundongos , RNA Guia de Cinetoplastídeos/metabolismo , Globinas beta/genética
5.
Sci Transl Med ; 13(591)2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33910976

RESUMO

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)-targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A-based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A-expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.


Assuntos
Hemoglobina Fetal , Receptores da Eritropoetina , Animais , Células Eritroides , Hemoglobina Fetal/genética , Macaca mulatta , Camundongos , Receptores da Eritropoetina/genética , Proteínas Repressoras
6.
Blood Adv ; 4(24): 6148-6156, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33351110

RESUMO

Intrabone (IB) injection of umbilical cord blood has been proposed as a potential mechanism to improve transplant engraftment and prevent graft failure. However, conventional IB techniques produce low retention of transplanted cells in the marrow. To overcome this barrier, we developed an optimized IB (OIB) injection method using low-volume, computer-controlled slow infusion that promotes cellular retention in the marrow. Here, we compare engraftment of CD34+ cells transplanted in a myeloablative rhesus macaque (RM) model using the OIB method compared with IV delivery. RM CD34+ cells obtained by apheresis were split equally for transduction with lentiviral vectors encoding either green fluorescent protein or yellow fluorescent protein reporters. Following conditioning, one marked autologous population of CD34+ cells was injected directly IB using the OIB method and the other was injected via slow IV push into the same animal (n = 3). Daily flow cytometry of blood quantified the proportion of engrafting cells deriving from each source. Marrow retention was examined using positron emission tomography/computed tomography imaging of 89Zirconium (89Zr)-oxine-labeled CD34+ cells. CD34+ cells injected via the OIB method were retained in the marrow and engrafted in all 3 animals. However, OIB-transplanted progenitor cells did not engraft any faster than those delivered IV and contributed significantly less to hematopoiesis than IV-delivered cells at all time points. Rigorous testing of our OIB delivery system in a competitive RM myeloablative transplant model showed no engraftment advantage over conventional IV infusion. Given the increased complexity and potential risks of IB vs IV approaches, our data do not support IB transplantation as a strategy to improve hematopoietic engraftment.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Animais , Antígenos CD34 , Macaca mulatta , Radioisótopos , Zircônio
7.
Blood Adv ; 4(23): 5976-5987, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33284949

RESUMO

Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation using a scalable, Good Manufacturing Practice-compliant system to restore protein expression and NADPH oxidase function. Dose-escalating clinical-scale in vivo studies in a nonhuman primate model verify the feasibility, safety, and persistence in peripheral blood of infusions of mRNA-transfected autologous granulocyte-enriched apheresis cells, supporting this novel therapeutic approach as a potential nonalloimmunizing adjunct treatment of intractable infections in CGD patients.


Assuntos
Remoção de Componentes Sanguíneos , Doença Granulomatosa Crônica , Granulócitos , Doença Granulomatosa Crônica/terapia , Humanos , NADPH Oxidases/genética , RNA Mensageiro/genética , Transfecção
8.
J Clin Invest ; 130(12): 6677-6687, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32897878

RESUMO

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer- or control locus-targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.


Assuntos
Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras , Animais , Humanos , Macaca mulatta , Camundongos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transplante Autólogo
9.
Nat Commun ; 10(1): 4479, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578323

RESUMO

Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level ß-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented ß-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and ß-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders.


Assuntos
Anemia Falciforme/terapia , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Globinas beta/genética , Anemia Falciforme/genética , Animais , Antígenos CD34/metabolismo , Vetores Genéticos/genética , Humanos , Macaca mulatta , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Reprodutibilidade dos Testes , Transplante Heterólogo , Globinas beta/metabolismo
10.
Mol Ther ; 27(9): 1586-1596, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31253582

RESUMO

Busulfan conditioning is utilized for hematopoietic stem cell (HSC) depletion in the context of HSC gene-therapy conditioning but may result in insufficient immunosuppression. In this study, we evaluated whether additional immunosuppression is required for efficient engraftment of gene-modified cells using a rhesus HSC lentiviral gene-therapy model. We transduced half of rhesus CD34+ cells with an enhanced green fluorescent protein (GFP)-encoding vector (immunogenic) and the other half with a γ-globin-encoding vector (no predicted immunogenicity). After autologous transplantation of both transduced cell populations following myeloablative busulfan conditioning (5.5 mg/kg/day for 4 days), we observed immunological rejection of GFP-transduced cells up to 3 months post-transplant and stable engraftment of γ-globin-transduced cells in two animals, demonstrating that ablative busulfan conditioning is sufficient for engraftment of gene-modified cells producing non-immunogenic proteins but insufficient to permit engraftment of immunogenic proteins. We then added immunosuppression with abatacept and sirolimus to busulfan conditioning and observed engraftment of both GFP- and γ-globin-transduced cells in two animals, demonstrating that additional immunosuppression allows for engraftment of gene-modified cells expressing immunogenic proteins. In conclusion, myeloablative busulfan conditioning should permit engraftment of gene-modified cells producing non-immunogenic proteins, while additional immunosuppression is required to prevent immunological rejection of a neoantigen.


Assuntos
Bussulfano/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Imunossupressores/farmacologia , Transgenes , Condicionamento Pré-Transplante , Animais , Expressão Gênica , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Macaca mulatta , Modelos Animais , Transdução Genética , gama-Globinas/genética
11.
Exp Hematol ; 75: 21-25.e1, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31173819

RESUMO

Elevated fetal hemoglobin (HbF) is associated with reduced severity of sickle cell disease. Therefore, γ-globin protein levels and F-cell (HbF-positive red blood cell) percentages are used for estimation of clinical benefit. Here, we monitored transplantation-related changes in HbF and F-cell percentages for rhesus macaques (Macaca mulatta) following total body irradiation or busulfan conditioning prior to CD34+ cell transplantation. HbF protein expression peaked in the first 4-9 weeks posttransplant (0.99%-2.53%), and F-cells increased in the first 6-17 weeks posttransplant (8.7%-45.3%). HbF and F-cell ratios gradually decreased and stabilized to levels similar to those of control animals (1.96 ± 1.97% for F cells and 0.49 ± 0.19% γ-globin expression) 4-7 months post-transplant. These findings confirm and expand on previous reports of transient induction in HbF and F-cell percentages in rhesus macaques following CD34+ cell transplantation, an observation that must be taken into consideration when evaluating therapeutic strategies that aim to specifically elevate HbF expression, which are currently in clinical development.


Assuntos
Anemia Falciforme/sangue , Anemia Falciforme/terapia , Eritrócitos Anormais/metabolismo , Hemoglobina Fetal/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Condicionamento Pré-Transplante , Aloenxertos , Animais , Antígenos CD34/metabolismo , Bussulfano/farmacologia , Eritrócitos Anormais/patologia , Macaca mulatta , Irradiação Corporal Total
12.
Hum Gene Ther Clin Dev ; 28(3): 136-144, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28447889

RESUMO

Steady state bone marrow (BM) is the preferred hematopoietic stem cell (HSC) source for gene therapy in sickle cell disease (SCD) due to the recognized risk of vaso-occlusive crisis during granulocyte colony-stimulating factor mobilization. We previously established clinically relevant HSC gene transfer in the rhesus model following transplantation of mobilized peripheral blood (PB) CD34+ cells transduced with lentiviral vectors. In this study, we examined steady state bone marrow (BM) in the rhesus competitive repopulation model and demonstrate similar gene marking in vitro and in vivo, as compared with mobilized PB CD34+ cells. We then evaluated PB and steady state BM in subjects with SCD and observed a higher frequency of CD34+ cells when compared with controls, likely due to enhanced hematopoiesis. However, CD34+ cell counts were reduced in both the PB and BM in patients treated with hydroxyurea, and hydroxyurea treatment strongly inhibited iPS cell generation from SCD subjects. Our data support that steady state BM is a useful HSC source for SCD gene therapy with similar transduction. The lower CD34+ percentages observed with hydroxyurea treatment warrants withholding hydroxyurea temporarily prior to harvesting HSCs. Our results are important for the design of gene targeting strategies for SCD.


Assuntos
Anemia Falciforme/sangue , Células da Medula Óssea/citologia , Terapia Genética/métodos , Células-Tronco Hematopoéticas/citologia , Anemia Falciforme/terapia , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Hematopoese , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Hidroxiureia/farmacologia , Macaca mulatta , Camundongos , Cultura Primária de Células/métodos
13.
Mol Ther Methods Clin Dev ; 3: 16059, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27652288

RESUMO

Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) gene therapy applications. However, low gene marking was previously observed in gene therapy trials, suggesting that RIC might be insufficient for (i) opening niches for efficient engraftment and/or (ii) inducing immunological tolerance for transgene-encoded proteins. Therefore, we evaluated both engraftment and tolerance for gene-modified cells using our rhesus HSC gene therapy model following RIC. We investigated a dose de-escalation of total body irradiation (TBI) from our standard dose of 10Gy (10, 8, 6, and 4Gy), in which rhesus CD34(+) cells were transduced with a VSVG-pseudotyped chimeric HIV-1 vector encoding enhanced green fluorescent protein (GFP) (or enhanced yellow fluorescent protein (YFP)). At ~6 months after transplantation, higher-dose TBI resulted in higher gene marking with logarithmic regression in peripheral blood cells. We then evaluated immunological tolerance for gene-modified cells, and found that lower-dose TBI allowed vigorous anti-GFP antibody production with logarithmic regression, while no significant anti-VSVG antibody formation was observed among all TBI groups. These data suggest that higher-dose TBI improves both engraftment and immunological tolerance for gene-modified cells. Additional immunosuppression might be required in RIC to induce tolerance for transgene products. Our findings should be valuable for developing conditioning regimens for HSC gene therapy applications.

14.
Blood ; 126(24): 2632-41, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26492933

RESUMO

Ionizing irradiation is used routinely to induce myeloablation and immunosuppression. However, it has not been possible to evaluate the extent of ablation without invasive biopsy. For lymphoid recovery, peripheral blood (PB) lymphocytes (PBLs) have been used for analysis, but they represent <2% of cells in lymphoid tissues (LTs). Using a combination of single-photon emission computed tomography imaging and a radiotracer ((99m)Tc-labeled rhesus immunoglobulin G1 anti-CD4R1 (Fab')2), we sequentially imaged CD4(+) cell recovery in rhesus macaques following total body irradiation (TBI) and reinfusion of vector-transduced, autologous CD34(+) cells. Our results present for the first time a sequential, real-time, noninvasive method to evaluate CD4(+) cell recovery. Importantly, despite myeloablation of circulating leukocytes following TBI, total depletion of CD4(+) lymphocytes in LTs such as the spleen is not achieved. The impact of TBI on LTs and PBLs is discordant, in which as few as 32.4% of CD4(+) cells were depleted from the spleen. In addition, despite full lymphocyte recovery in the spleen and PB, lymph nodes have suboptimal recovery. This highlights concerns about residual disease, endogenous contributions to recovery, and residual LT damage following ionizing irradiation. Such methodologies also have direct application to immunosuppressive therapy and other immunosuppressive disorders, such as those associated with viral monitoring.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Tecido Linfoide/fisiologia , Tomografia Computadorizada de Emissão de Fóton Único , Condicionamento Pré-Transplante , Animais , Medula Óssea/efeitos da radiação , Antígenos CD4/genética , Contagem de Linfócito CD4 , Sistemas Computacionais , Genes Reporter , Genes Sintéticos , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Imunoglobulina G/genética , Lentivirus/genética , Linfonodos/imunologia , Linfonodos/efeitos da radiação , Tecido Linfoide/diagnóstico por imagem , Tecido Linfoide/efeitos da radiação , Macaca mulatta , Imagem Multimodal , Especificidade de Órgãos , Quimera por Radiação , Baço/imunologia , Baço/efeitos da radiação , Tomografia Computadorizada por Raios X , Transdução Genética , Transplante Autólogo , Irradiação Corporal Total
15.
Cell Stem Cell ; 14(4): 473-85, 2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24702996

RESUMO

In mice, clonal tracking of hematopoietic stem cells (HSCs) has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3-10 years), arising in sequential groups and likely representing self-renewing HSCs. The remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid, or both myeloid and lymphoid lineages. Over time, the 4%-10% of clones with robust dual lineage contribution predominated in repopulation. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically relevant model over a long time frame and provides a substantial system-level data set that is a reference point for future work.


Assuntos
Diferenciação Celular , Linhagem da Célula , Rastreamento de Células , Células-Tronco Hematopoéticas/citologia , Linfócitos/citologia , Células Mieloides/citologia , Animais , Células Cultivadas , Vetores Genéticos , Células-Tronco Hematopoéticas/metabolismo , Linfócitos/metabolismo , Macaca mulatta , Camundongos , Células Mieloides/metabolismo , RNA Interferente Pequeno/genética , Receptores CCR5/química , Receptores CCR5/genética , Receptores CCR5/metabolismo , Transgenes/fisiologia
16.
Mol Ther Nucleic Acids ; 2: e122, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24045711

RESUMO

Hematopoietic stem cell (HSC) gene therapy using integrating vectors has a potential leukemogenic risk due to insertional mutagenesis. To reduce this risk, a limitation of ≤2 average vector copy number (VCN) per cell is generally accepted. We developed an assay for VCN among transduced CD34(+) cells that reliably predicts in vivo VCN in 16 rhesus recipients of CD34(+) cells transduced with a green fluorescent protein (GFP) (or yellow fluorescent protein (YFP))-encoding lentiviral vector. Using GFP (or YFP)-specific probe/primers by real-time PCR, VCN among transduced CD34(+) cells had no correlation with VCN among granulocytes or lymphocytes in vivo assayed 6 months post-transplantation. This was a likely result of residual plasmids present in the vector preparation. We then designed self-inactivating long terminal repeat (SIN-LTR)-specific probe/primers, which detect only integrated provirus. Evaluation with SIN-LTR probe/primers resulted in a positive correlation of VCN among transduced CD34(+) cells with granulocytes and lymphocytes in vivo. The transduced CD34(+) cells had higher VCN (25.1 ± 5.6) as compared with granulocytes (2.8 ± 1) and lymphocytes (2.4 ± 0.7). In summary, an integrated provirus-specific real-time PCR system demonstrated nine- to tenfold higher VCN in transduced CD34(+) cells in vitro, as compared with VCN in vivo. Therefore, the restriction of ≤2 VCN before infusion might unnecessarily limit gene transfer efficacy.Molecular Therapy-Nucleic Acids (2013) 2, e122; doi:10.1038/mtna.2013.49; published online 17 September 2013.

17.
Mol Ther ; 20(10): 1882-92, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22871664

RESUMO

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoformα (TRIM5α) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (χHIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34(+) cells. To evaluate whether χHIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34(+) cells were transduced with standard SIV vectors and the other half with χHIV vectors. As compared with SIV vectors, χHIV vectors achieved higher vector integration, and the transgene expression rates were two- to threefold higher in granulocytes and red blood cells and equivalent in lymphocytes and platelets for 2 years. A recipient of χHIV vector-only transduced cells reached up to 40% of transgene expression rates in granulocytes and lymphocytes and 20% in red blood cells. Similar to HIV1 and SIV vectors, χHIV vector frequently integrated into gene regions, especially into introns. In summary, our χHIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This χHIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models.


Assuntos
Vetores Genéticos/genética , HIV-1/genética , Células-Tronco Hematopoéticas , Transdução Genética , Animais , Antígenos CD34/metabolismo , Southern Blotting , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Transplante de Células-Tronco Hematopoéticas , Humanos , Macaca mulatta , Reação em Cadeia da Polimerase em Tempo Real , Vírus da Imunodeficiência Símia/genética , Transgenes
18.
Blood ; 118(25): 6580-90, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21989987

RESUMO

In this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4(+)/CD25(high)/CD127(low)/FoxP3(+) Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8(+) T cells were mobilized to a greater extent than CD4(+) T cells, with accumulation of 3.7 ± 0.4-fold more total CD8+ T cells and 6.2 ± 0.4-fold more CD8(+) effector memory T cells in the leukapheresis product compared with G-CSF alone. Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation.


Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Compostos Heterocíclicos/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Benzilaminas , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Ciclamos , Sinergismo Farmacológico , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Leucaférese/métodos , Contagem de Linfócitos , Macaca mulatta , Receptores CXCR4/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo
19.
Cell Stem Cell ; 8(4): 445-58, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21474107

RESUMO

Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34(+) hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34(+) MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.


Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Dinoprostona/farmacologia , Modelos Animais , Animais , Antígenos CD34/análise , Ensaio de Unidades Formadoras de Colônias , Dinoprostona/análogos & derivados , Sobrevivência de Enxerto , Humanos , Primatas , Transplante Heterólogo
20.
J Virol ; 84(22): 11771-80, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20844053

RESUMO

Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5' and the 3' vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.


Assuntos
Células-Tronco Hematopoéticas/virologia , Ensaios de Triagem em Larga Escala/métodos , Lentivirus/fisiologia , Análise de Sequência de DNA/métodos , Integração Viral , Animais , Células Cultivadas , Células Clonais , Terapia Genética , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Células-Tronco Hematopoéticas/citologia , Lentivirus/genética , Macaca mulatta
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