In vivo and ex vivo gene therapy strategies to treat tumors using adenovirus gene transfer vectors

1999 ◽  
Vol 43 (7) ◽  
pp. S90-S99 ◽  
Author(s):  
Ronald G. Crystal
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Ex Vivo Gene Therapy ◽  
Transfer Vectors ◽  
Therapy Strategies

scholarly journals Keratinocyte Gene Transfer and Gene Therapy

1996 ◽  
Vol 7 (3) ◽  
pp. 204-221 ◽  
Author(s):  
J.A. Garlick ◽  
E.S. Fenjves
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Target Cells ◽  
Gene Products ◽  
Oral Keratinocytes ◽  
Systemic Delivery ◽  
Therapy Strategies

Gene therapy has moved beyond the pre-clinical stage to the treatment of a variety of inherited and acquired diseases. For such therapy to be successful, genes must be efficiently delivered to target cells and gene products must be expressed for prolonged periods of time without toxic effects to the host. This may be achieved by means of an in vivo strategy where genes are transferred directly into a host cell, or by means of an ex vivo approach through which cells are removed, cultured, targeted for gene delivery, and grafted back to the host. Several obstacles continue to delay safe and effective clinical application of gene therapy in a variety of target cells. The limited survival of transplanted cells, transient expression of transferred genes, and difficulties in targeting stem cells are technical issues requiring further investigation. Epidermal and oral keratinocytes are potential vehicles for gene therapy. Several features of these tissues can be utilized to achieve delivery of therapeutic gene products for local or systemic delivery. These qualities include: (1) the presence of stem cells; (2) the cell-, strata-, and site-specific regulation of keratinocyte gene expression; (3) tissue accessibility; and (4) secretory capacity. Such features can be exploited by the use of gene therapy strategies to facilitate: (1) identification, enrichment, and targeting of stem cells to ensure the continued presence of the transferred gene; (2) high-level and persistent transgene expression using keratinocyte-specific promoters; (3) tissue access needed for culture and grafting for ex vivo therapy and direct in vivo gene transfer; (4) secretion of transgene product for local or systemic delivery; and (5) monitoring of genetically modified tissue and removal if treatment termination is required. Optimal gene therapy strategies are being tested in a variety of tissues to treat dominant and recessive genetic disorders as well as acquired diseases such as neoplasia and infectious disease. This experience provides a basis for the application of such clinical studies to a spectrum of diseases effecting epidermal and oral keratinocytes. Gene therapy is in an early stage yet holds great promise for its ultimate clinical application.

scholarly journals Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model

2021 ◽  
Vol 18 ◽  
pp. 347-354
Author(s):  
Masashi Noda ◽  
Kohei Tatsumi ◽  
Hideto Matsui ◽  
Yasunori Matsunari ◽  
Takeshi Sato ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Canine Model ◽  
Gene Transfer Techniques

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 Ex Vivo and In Vivo Adenovirus-Mediated Gene Therapy Strategies Induce a Systemic Anti-Tumor Immune Defence in the B16 Melanoma Model

1998 ◽  
Vol 110 (6) ◽  
pp. 867-871 ◽  
Author(s):  
Bernd Bonnekoh ◽  
David A. Greenhalgh ◽  
Shu-Hsia Chen ◽  
Andreas Block ◽  
Susan S. Rich ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Ex Vivo ◽  
B16 Melanoma ◽  
Immune Defence ◽  
Melanoma Model ◽  
Therapy Strategies

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.

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.

Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)–deficient mice and corrects their immune and metabolic defects

Blood ◽  
2006 ◽  
Vol 108 (9) ◽  
pp. 2979-2988 ◽  
Author(s):  
Alessandra Mortellaro ◽  
Raisa Jofra Hernandez ◽  
Matteo M. Guerrini ◽  
Filippo Carlucci ◽  
Antonella Tabucchi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Adenosine Deaminase ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Lentiviral Vectors ◽  
Mixed Chimerism ◽  
Pulmonary Insufficiency ◽  
Ex Vivo Gene Therapy

AbstractAdenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA–/– mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach.

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.

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