scholarly journals Engraftment and in vivo proliferation advantage of gene-corrected mobilized CD34+ cells from Fanconi anemia patients

Blood ◽  
2017 ◽  
Vol 130 (13) ◽  
pp. 1535-1542 ◽  
Author(s):  
Paula Río ◽  
Susana Navarro ◽  
Guillermo Guenechea ◽  
Rebeca Sánchez-Domínguez ◽  
Maria Luisa Lamana ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Fanconi Anemia ◽  
Ex Vivo ◽  
Cd34 Cells ◽  
Key Points ◽  
Ex Vivo Gene Therapy

Key Points First evidence of phenotypic correction in FA hematopoietic repopulating cells by optimized collection and short transduction of CD34+ cells. Optimized ex vivo gene therapy of FA CD34+ cells confers proliferation advantage to phenotypically corrected repopulating cells.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

scholarly journals Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Blood ◽  
2018 ◽  
Vol 131 (26) ◽  
pp. 2915-2928 ◽  
Author(s):  
Chang Li ◽  
Nikoletta Psatha ◽  
Pavel Sova ◽  
Sucheol Gil ◽  
Hongjie Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Binding Site ◽  
Genome Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Cell Genome ◽  
Hsc Transplantation

Key Points CRISPR/Cas9-mediated disruption of a BCL11A binding site in HSCs of β-YAC mice results in the reactivation of γ-globin in erythrocytes. Our approach for in vivo HSC genome editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.

Rescue of Lethal Hypophosphatasia Mice by Neonatal Ex Vivo Gene Therapy Using Lentivirally Transduced Bone Marrow Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3154-3154
Author(s):  
Osamu Iijima ◽  
Koichi Miyake ◽  
Hanako Sugano-Tajima ◽  
Tsutomu Igarashi ◽  
Chizu Kanokoda ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Bone Mineralization ◽  
Epileptic Seizures ◽  
Alp Activity ◽  
Donor Cells ◽  
Ex Vivo Gene Therapy

Abstract Abstract 3154 Hypophosphatasia (HPP) is an inherited skeletal disease caused by genetic defects of tissue-nonspecific alkaline phosphatase (TNALP). TNALP is an ectoenzyme which is attached to the outside plasma membrane via a GPI anchor and plays an essential role in bone mineralization. The major symptoms are hypomineralization of systemic bones, respiratory insufficiency and epileptic seizures. Severe HPP is often fatal. Since ALP functions on the exterior of the cells, enzyme replacement therapy (ERT) is a potential approach to treat HPP. Although previous trials of ERT using various forms of soluble ALP showed no clinical benefit, it was recently demonstrated that TNALP with deca-aspartates at the C terminus (TNALP-D10) had a high affinity for bone tissue and repeated injections of TNALP-D10 successfully rescued lethal HPP mice. HPP mice were generated by knockout the mouse TNALP gene (Akp2) and phenotypically mimic to severe infantile HPP and develop hypomineralization, growth failure and epileptic seizures after birth. The plasma ALP activity in HPP mice was less than 0.01 U/ml (approx. 0.1 U/ml in wild type (wt) mice) and the average life span of non-treated HPP mice is about 20 days. We have also shown that a single intravenous injection of either lentiviral or AAV vector expressing TNALP-D10 resulted in prolonged survival and phenotypic correction of HPP mice. In this in vivo gene therapy, bone cells were not efficiently transduced, but the plasma ALP activity derived from TNALP-D10 secreted from transduced liver or muscle cells was maintained at extremely high levels (10 to 100 folds higher than that of wt mice). As an alternative approach, we are studying the feasibility of hematopoietic stem cells (HSC) based ex vivo gene therapy for HPP. After homing of HSC to the bone marrow, local expression of TNALP in the bone should be beneficial to improve bone mineralization. Other potential advantages of this strategy compared with an in vivo systemic gene therapy include lifelong expression of TNALP, no risk of germline gene transfer, and no immunoreaction against viral vector. Lineage negative bone marrow cells (BMC) were harvested from B6.CD45.1 mice (Ly5.1) using the Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD Bioscience) and incubated with lentiviral vector expressing GFP or TNALP-D10 for 20 hrs at an moi of 50 with mSCF, mIL3 and rhIL6. Transduction efficiency assessed by GFP expression was approximately 40 % under the condition used. Recipient neonatal mice (Ly5.2) were sub-lethally irradiated at 4Gy and received BMC (1 × 106̂ cells) through the jugular vein on day 2. Irradiated neonatal wt mice showed a slight reduction of the growth rate but normal physical activity and healthy appearance. GFP positive donor cells migrated to the bone marrow in recipient mice. FACS analysis of the peripheral blood samples 4 to 12 weeks after transplantation demonstrated that approximately 30 % of Ly5.1 donor cells were stably detected in all lineage blood cells of recipient mice. After treatment of neonatal HPP mice with TNALP-D10 expressing BMC, the plasma ALP activity was elevated to 1 to 2 U/ml at 4 weeks of age and remained at this level during the observation period. The treated mice actively moved in the cage without epileptic seizures and the life span was prolonged over 3 months. X-ray examination of the skeleton showed that mineralization was significantly improved compared to non-treated HPP mice, but not completely normalized compared to age matched wt mice. These results indicate that lentivirally transduced BMC can serve as a reservoir for continuous supply of TNALP-D10 to rescue lethal HPP mice. However, the concentration of TNALP-D10 in the bone may not be sufficient for complete correction of skeletal abnormalities. Further optimization of gene transfer and neonatal BMT is under way to increase the plasma ALP activity. HSC mediated ex vivo gene therapy is now being applied to treat not only hematological diseases but also neurological disorders such as adreno leukodystrophy and metachromatic leukodystrophy. Hypophosphatasia, a systemic bone disease, is also an important target for ex vivo gene therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gene Therapy for Neuronopathic Mucopolysaccharidoses: State of the Art

2021 ◽  
Vol 22 (17) ◽  
pp. 9200
Author(s):  
María José de Castro ◽  
Mireia del Toro ◽  
Roberto Giugliani ◽  
María Luz Couce
Keyword(s):  
Gene Therapy ◽  
State Of The Art ◽  
Ex Vivo ◽  
Specific Treatment ◽  
Medical Community ◽  
Major Step ◽  
Enzyme Replacement ◽  
Ex Vivo Gene Therapy ◽  
The Central Nervous System

The need for long-lasting and transformative therapies for mucopolysaccharidoses (MPS) cannot be understated. Currently, many forms of MPS lack a specific treatment and in other cases available therapies, such as enzyme replacement therapy (ERT), do not reach important areas such as the central nervous system (CNS). The advent of newborn screening procedures represents a major step forward in early identification and treatment of individuals with MPS. However, the treatment of brain disease in neuronopathic MPS has been a major challenge to date, mainly because the blood brain barrier (BBB) prevents penetration of the brain by large molecules, including enzymes. Over the last years several novel experimental therapies for neuronopathic MPS have been investigated. Gene therapy and gene editing constitute potentially curative treatments. However, despite recent progress in the field, several considerations should be taken into account. This review focuses on the state of the art of in vivo and ex vivo gene therapy-based approaches targeting the CNS in neuronopathic MPS, discusses clinical trials conducted to date, and provides a vision for the future implications of these therapies for the medical community. Recent advances in the field, as well as limitations relating to efficacy, potential toxicity, and immunogenicity, are also discussed.

Methylguanine Methyltransferase-Based In Vivo Selection Results in Only Transient Improvement in Long-Term Marking after Autologous Transplantation of Transduced Hematopoietic Stem Cells in Rhesus Macaques.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3272-3272
Author(s):  
Andre Larochelle ◽  
Uimook Choi ◽  
Nora Naumann ◽  
Josh R. Clevenger ◽  
Harry L. Malech ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Steady State ◽  
Ex Vivo ◽  
Rhesus Macaques ◽  
Autologous Transplantation ◽  
Survival Advantage ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Selective Survival

Abstract In vivo selective survival advantage of transduced cells contributed to clinically beneficial levels of genetic correction of lymphocytes following X-SCID gene therapy. For most blood disorders there will be no constitutive selective advantage of the gene-corrected cells. Alternatively, a selectable gene incorporated into the vector may provide selective survival advantage. The P140K mutant of human O6-methylguanine-DNA methyltransferase (MGMT*) is a candidate mammalian selectable gene for hematopoietic stem cell (HSC) gene therapy. AMD3100-mobilized CD34+ cells from 5 rhesus macaques were transduced daily from day 2 to 4 of culture using oncoretroviral (n=2 animals) or lentiviral (n=3 animals) vectors encoding the gp91phox-IRES-MGMT* cassette or the GFP-MGMT* fusion protein, respectively. Transduced CD34+ cells were selected after (in vivo, n=4) or before (ex vivo, n=1) autologous transplantation in rhesus macaques using the BG (120mg/m2)/TMZ 400 mg/m2 combination for in vivo selection and the BG (5uM)/BCNU (7.5uM) combination for ex vivo selection. Marking of peripheral blood (PB) cells was evaluated by FACS and/or real-time PCR. Bulk CD34+ cells were marked at 27–58% after transduction with oncoretroviral or lentiviral vectors. Four animals were transplanted with transduced non-selected CD34+ cells. Small fractions of cultured cells not transplanted were exposed to BG/BCNU resulting in an increase of marking to 88–97% in each case, confirming the in vitro survival advantage. Cells from animals #1 and #2 were transduced with oncoretroviral vectors and steady-state marking of 3.5% was obtained in PB. Animal #1 received BG/TMZ infusions at 3 and 6 months post-transplant. Marking declined to 3.3% and 1.1% after BG/TMZ treatment 1 and 2, respectively. Animal #2 received one cycle of BG/TMZ at 4 months post-transplant. Full hematopoietic recovery was not achieved and the animal died of infectious complications one month after treatment. Marking of 2% was detected in the PB at the time of death. Cells from animals #3 and #4 were transduced with lentiviral vectors. Animal #3 received 4 monthly infusions of BG/TMZ starting 5 months after transplantation. Marking increased from 0.1% at steady-state to 1.8% in PB after the first cycle but rapidly declined to 0.2%. Despite significant myelosuppression, additional cycles of BG/TMZ resulted in no significant improvement in marking. Animal #4 received 4 monthly infusions of BG/TMZ starting 3 months after transplantation. Marking increased from 3.3% at steady-state to 29.2% after the first cycle but rapidly declined to 6.2%. Each additional cycle of BG/TMZ resulted in a transient increase in marking with a peak increase gradually declining with each cycle. Animal #5 was transplanted with CD34+ cells transduced with lentiviral vector expressing GFP-MGMT* and exposed to BG/BCNU ex vivo before transplantation. At the time of reinfusion, 55% of the cells were vector positive. Stable hematopoietic recovery required one month, compared to an average recovery of 2 weeks in animals transplanted with transduced cells without ex vivo selection. Steady state marking in PB of only 0.7% was detected. These data combined with the theoretic concern that the use of cytotoxic drugs could increase the risk of leukemogenesis in the setting of drug-resistance gene therapy, raise concerns for the clinical applicability of this approach.

Bone Marrow Cell Based Enzyme Replacement Prolongs Survival and Improves Disease Phenotypes In a Mouse Model Of Lethal Hypophosphatasia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2897-2897 ◽  
Author(s):  
Osamu Iijima ◽  
Koichi Miyake ◽  
Aki Nakamura ◽  
Tsutomu Igarashi ◽  
Chizu Kanokoda ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Viral Vector ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Bone Mineralization ◽  
Epileptic Seizures ◽  
Enzyme Replacement ◽  
Alp Activity ◽  
Ex Vivo Gene Therapy

Abstract Hypophosphatasia (HPP) is an inherited skeletal disease caused by mutations of the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNALP). TNALP is an ectoenzyme and plays an essential role in bone mineralization. The major symptoms of HPP are hypomineralization of systemic bones, respiratory insufficiency and epileptic seizures. Perinatal and infantile forms of HPP are often fatal. Since ALP functions on the exterior of the cells, enzyme replacement therapy (ERT) is a potential approach to treat HPP. Currently, Phase II/III clinical trials of ERT using a recombinant TNALP which linked deca-aspartate (D10) at the C terminus for bone targeting are ongoing in North America, Europe and Japan. The perinatal and infantile patients received the ERT showed apparent improvement of the symptoms. However, the ERT is highly invasive for the young patients because it requires repeated subcutaneous administration of large amounts of the enzyme every 3 times a week for long-term correction. As another approach to treat HPP, we have reported in vivo gene therapy for ALPL (Akp2) knock-out mice (HPP mice). The treated HPP mice were rescued by a single systemic injection of lentiviral vector or adeno-associated viral vector expressing bone targeted form of TNALP (TNALP-D10) during the neonatal or fetal period. Although untreated HPP mice developed apparent growth failure and died by around 20 days of age due to severe skeletal hypomineralization and epileptic seizure, the treated HPP mice were prolonged the survival and improved the physical activity. In the treated HPP mice, plasma ALP activity was kept higher than 1 U/ml (approximately 0.01 U/ml in untreated HPP mice and 0.1 U/ml in wild type (WT) mice) which gives therapeutic effects. However, disadvantages of in vivo gene therapy include the risk of germline gene transfer and induction of immune responses to the vectors or transgene products. To overcome these problems, we examined a feasibility of ex vivo gene therapy using hematopoietic stem cells (HSC) transduced by lentiviral vector expressing TNALP-D10. The potential advantages of this approach are lifelong expression of TNALP-D10 and prevention of risks of in vivo gene therapy. The lineage negative bone marrow cells containing HSC (Lin- BMC) were harvested from B6.CD45.1 mice (Ly5.1) and then enriched using Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD bioscience). Lin- BMC was transduced with lentiviral vector expressing TNALP-D10 for 20 hrs at an moi of 50 with mSCF, mIL3 and rhIL6 on Retronectin coated plate. Recipient HPP mice (Ly5.2) on day 2 after birth were received a sub-lethal dose of total body irradiation (4Gy) 4hr prior to transplantation. Then, the transduced Lin- BMC (1 x 106 cells) was transplanted intravenously into the HPP mice through the temporal vein or jugular vein. The plasma ALP activity was rapidly elevated approximately 400 fold higher than untreated HPP mice (untreated: 0.014±0.004 units/ml (n=4) and treated: 5.39±2.29 units/ml (n=7), respectively) on 1 week after the transplantation and kept at this level during the observation period. Engraftment rate of Ly5.1 donor cells were sustained at approximately 30-40% with multilineage potential. The treated HPP mice were prolonged their survival over 3 months without epileptic seizures and the physical activities were improved. The histochemical ALP staining indicated TNALP-D10 was accumulated on the surface of trabecular and cortical bones of the treated HPP mice. The bone mineralization was significantly improved, but still not satisfactory compared with age matched WT mice. Contrary to our expectations, 2 of 9 HPP mice transplanted with non-transduced BMC also survived for 3 months. However, the plasma ALP activity was not elevated at all and the bone mineralization was incomplete compared with treated HPP mice. These results indicate that a single transplantation of genetically modified BMC at neonatal period is sufficient for long-term supply of TNALP-D10 and rescue of lethal HPP mice, even though hypomineralization was not completely recovered. Further optimization of viral vector and conditioning of transplantation is required to increase the treatment efficacy for HPP. However, neonatal ex vivo gene therapy using genetically modified BMC would be a possible and practical approach to treat HPP. Disclosures: Watanabe: Alexion Pharmaceuticals, Inc.: Membership on an entity’s Board of Directors or advisory committees.

CRISPR/Cas9 system: a powerful technology for in vivo and ex vivo gene therapy

2017 ◽  
Vol 60 (5) ◽  
pp. 468-475 ◽  
Author(s):  
Xiaohui Zhang ◽  
Liren Wang ◽  
Mingyao Liu ◽  
Dali Li
Keyword(s):  
Gene Therapy ◽  
Ex Vivo ◽  
System A ◽  
Ex Vivo Gene Therapy

Use of bone morphogenetic protein—9 gene therapy to induce spinal arthrodesis in the rodent

2000 ◽  
Vol 92 (2) ◽  
pp. 191-196 ◽  
Author(s):  
Gregory A. Helm ◽  
Tord D. Alden ◽  
Elisa J. Beres ◽  
Sarah B. Hudson ◽  
Subinoy Das ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Morphogenetic Proteins ◽  
Ex Vivo ◽  
Content Type ◽  
Ex Vivo Gene Therapy ◽  
Bone Morphogenetic Protein 9 ◽  
Spinal Arthrodesis ◽  
Fine Print

Object. Bone morphogenetic proteins (BMPs) have been shown to have significant osteoinductive activity in numerous in vitro and in vivo assay systems, and BMP-2 and BMP-7 are currently being evaluated in human clinical studies. In the spinal region, BMPs have been shown to promote spinal arthrodesis at a higher rate than autologous bone alone. The delivery of BMPs via direct or ex vivo gene therapy techniques is also currently being evaluated and has shown promise in several mammalian models. The present study was designed to evaluate the efficacy of the use of direct, percutaneous BMP-9 adenoviral gene therapy to promote spinal fusion in the rodent. Methods. Each animal was injected with 7.5 × 108 pfu of a BMP-9 adenoviral vector in the lumbar paraspinal musculature and allowed to survive 16 weeks. Computerized tomography studies and histological analysis demonstrated massive bone induction at the injection sites, clearly leading to solid spinal arthrodesis, without evidence of pseudarthroses, nerve root compression, or systemic side effects. Conclusions. The results of this study strongly support the advancement of BMP gene therapy techniques toward clinical use.

scholarly journals Cell and Gene Therapy for Anemia: Hematopoietic Stem Cells and Gene Editing

2021 ◽  
Vol 22 (12) ◽  
pp. 6275
Author(s):  
Dito Anurogo ◽  
Nova Yuli Prasetyo Budi ◽  
Mai-Huong Thi Ngo ◽  
Yen-Hua Huang ◽  
Jeanne Adiwinata Pawitan
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Ex Vivo ◽  
Risk Mitigation ◽  
Future Research ◽  
Hematopoietic Stem ◽  
Cell And Gene Therapy

Hereditary anemia has various manifestations, such as sickle cell disease (SCD), Fanconi anemia, glucose-6-phosphate dehydrogenase deficiency (G6PDD), and thalassemia. The available management strategies for these disorders are still unsatisfactory and do not eliminate the main causes. As genetic aberrations are the main causes of all forms of hereditary anemia, the optimal approach involves repairing the defective gene, possibly through the transplantation of normal hematopoietic stem cells (HSCs) from a normal matching donor or through gene therapy approaches (either in vivo or ex vivo) to correct the patient’s HSCs. To clearly illustrate the importance of cell and gene therapy in hereditary anemia, this paper provides a review of the genetic aberration, epidemiology, clinical features, current management, and cell and gene therapy endeavors related to SCD, thalassemia, Fanconi anemia, and G6PDD. Moreover, we expound the future research direction of HSC derivation from induced pluripotent stem cells (iPSCs), strategies to edit HSCs, gene therapy risk mitigation, and their clinical perspectives. In conclusion, gene-corrected hematopoietic stem cell transplantation has promising outcomes for SCD, Fanconi anemia, and thalassemia, and it may overcome the limitation of the source of allogenic bone marrow transplantation.

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