No lung toxicity after repeated aerosol or intravenous delivery of plasmid-cationic liposome complexes

1994 ◽  
Vol 77 (1) ◽  
pp. 415-419 ◽  
Author(s):  
A. E. Canonico ◽  
J. D. Plitman ◽  
J. T. Conary ◽  
B. O. Meyrick ◽  
K. L. Brigham
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Delivery System ◽  
Cationic Liposome ◽  
Cationic Liposomes ◽  
Lung Compliance ◽  
Arterial Oxygen ◽  
Ideal System ◽  
Alpha 1 Antitrypsin

The safety aspects of human gene therapy are of paramount importance in developing an ideal system for gene transfer. Lipofection using DNA in the form of a plasmid has been shown to successfully transfect the lungs when administered either intravenously or by aerosol. We have shown that repeated intravenous or aerosol administration of a plasmid containing the recombinant human alpha 1-antitrypsin gene and a cytomegalovirus promoter complexed to cationic liposomes results in no adverse effects on pulmonary histology, lung compliance, lung resistance, or alveolar-arterial oxygen gradient. Immunohistochemistry and Western blot analysis confirm successful gene transfer using this delivery system. We conclude that plasmids complexed to cationic liposomes may be a safe and efficacious delivery system for in vivo gene transfer to the lungs. Using this delivery system, in vivo gene therapy to the lungs can be achieved by either intravenous or aerosol administration of the transgene.

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

scholarly journals Essential Role of Host Double-Stranded DNA Released from Dying Cells by Cationic Liposomes for Mucosal Adjuvanticity

Vaccines ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 8
Author(s):  
Rui Tada ◽  
Akihiro Ohshima ◽  
Yuya Tanazawa ◽  
Akari Ohmi ◽  
Saeko Takahashi ◽  
...  
Keyword(s):  
Essential Role ◽  
Cationic Liposome ◽  
Cationic Liposomes ◽  
Mucosal Vaccine ◽  
Nasal Administration ◽  
Adjuvant Activity ◽  
Mucosal Adjuvant ◽  
Double Stranded Dna ◽  
Dying Cells

Infectious disease remains a substantial cause of death. To overcome this issue, mucosal vaccine systems are considered to be a promising strategy. Yet, none are approved for clinical use, except for live-attenuated mucosal vaccines, mainly owing to the lack of effective and safe systems to induce antigen-specific immune responses in the mucosal compartment. We have reported that intranasal vaccination of an antigenic protein, with cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl], induced antigen-specific mucosal and systemic antibody responses in mice. However, precise molecular mechanism(s) underlying the mucosal adjuvant effects of cationic liposomes remain to be uncovered. Here, we show that a host double-stranded DNA (dsDNA), released at the site of cationic liposome injection, plays an essential role for the mucosal adjuvanticity of the cationic liposome. Namely, we found that nasal administration of the cationic liposomes induced localized cell death, at the site of injection, resulting in extracellular leakage of host dsDNA. Additionally, in vivo DNase I treatment markedly impaired OVA-specific mucosal and systemic antibody production exerted by cationic liposomes. Our report reveals that host dsDNA, released from local dying cells, acts as a damage-associated molecular pattern that mediates the mucosal adjuvant activity of cationic liposomes.

Efficacy of cationic liposome-mediated gene transfer to mesangial cells in vitro and in vivo

2001 ◽  
Vol 79 (4) ◽  
pp. 184-189 ◽  
Author(s):  
Henning Madry ◽  
Regina Reszka ◽  
Jürgen Bohlender ◽  
Jürgen Wagner
Keyword(s):  
Gene Transfer ◽  
Mesangial Cells ◽  
Cationic Liposome

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.

Phenotypic Correction of Adamts13-Deficient Mice by Early Intra-Amniotic Gene Transfer of Lentiviral Vector Encoding ADAMTS13 Genes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 197-197
Author(s):  
Masami Niiya ◽  
Masayuki Endo ◽  
Philip W. Zoltick ◽  
Nidal E. Muvarak ◽  
David G. Motto ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Proteolytic Activity ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Occlusion Time ◽  
Carboxyl Terminal ◽  
Human Wild Type

Abstract ADAMTS13, a member of A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, is mainly synthesized in the hepatic stellate cells, endothelial cells and megakaryocytes or platelets. It controls the sizes of von Willebrand factor (VWF) multimers by cleaving VWF at the Tyr1605-Met1606 bond. Genetic deficiency of plasma ADAMTS13 activity results in hereditary thrombotic thrombocytopenic purpura (TTP), also named Upshaw-Schülman syndrome. To develop a potential gene therapy approach and to determine the domains of ADAMTS13 required for recognition and cleavage of VWF in vivo, a self-inactivating lentiviral vector encoding human wild-type ADAMTS13 or variant truncated after the spacer domain (construct MDTCS) was administrated by intra-amniotic injection on embryonic day 8. Direct stereomicroscopy and immunofluorescent microscopic analysis revealed that the green fluorescent protein (GFP) reporter, ADAMTS13 and MDTCS were predominantly expressed in the heart, kidneys and skin. The synthesized ADAMTS13 and truncated variant were detectable in mouse plasma by immunoprecipitation and Western blot, as well as by proteolytic cleavage of FRETS-VWF73 substrate. The levels of proteolytic activity in plasma of mice expressing ADAMTS13 and MDTCS were 5 ± 7% and 60 ± 70%, respectively using normal human plasma as a standard, and this proteolytic activity persisted for at least 24 weeks in Adamts13−/−mice and 42 weeks in wild-type mice tested (the duration of observation). The mice expressing both recombinant ADAMTS13 and MDTCS showed a significantly decreased ratio of plasma VWF collagen-binding activity to antigen and a reduction in VWF multimer sizes as compared to those in the controls. Moreover, the mice expressing ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time (9.0 ± 0.6 and 25.2 ± 3.2 min, respectively) as compared to the Adamts13−/− mice expressing GFP alone (5.6 ± 0.5 min) (p&lt;0.01). The ferric chloride-induced carotid occlusion time in Adamts13−/− mice expressing ADAMTS13 was almost identical to that in wild type mice with same genetic background (C56BL/6) (8.0 ± 0.2 min) (p&gt;0.05). The data demonstrate the correction of the prothrombotic phenotype in Adamts13−/−mice by gene transfer to the fetus by viral vectors encoding human wild type ADAMTS13 and the carboxyl terminal truncated variant (MDTCS), supporting the feasibility of developing a gene therapy based treatment for hereditary TTP. The discrepancy in the proteolytic activity of MDTCS between in vitro (Zhang P et al. Blood, 2007 in press) and in vivo in the present study suggests the potential cofactors in murine circulation that may rescue the defective proteolytic activity of the carboxyl-terminal truncated ADAMTS13 protease seen in vitro.

The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2839-2848 ◽  
Author(s):  
Hitoshi Hibino ◽  
Kenzaburo Tani ◽  
Kenji Ikebuchi ◽  
Ming-Shiuan Wu ◽  
Hajime Sugiyama ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Nonhuman Primate ◽  
Peripheral Blood ◽  
Common Marmoset ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
The Common

Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (<1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting hematopoietic stem cells.

Neonatal or hepatocyte growth factor–potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3924-3932 ◽  
Author(s):  
Lingfei Xu ◽  
Cuihua Gao ◽  
Mark S. Sands ◽  
Shi-Rong Cai ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Growth Factor ◽  
Gene Transfer ◽  
Hepatocyte Growth Factor ◽  
Retroviral Vector ◽  
Factor Ix ◽  
Hemophilia B ◽  
Hepatocyte Growth

AbstractHemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus–based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 μg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.

scholarly journals Wild-type p53 gene transfer is not detrimental to normal cells in vivo: implications for tumor gene therapy

Oncogene ◽  
2004 ◽  
Vol 23 (2) ◽  
pp. 418-425 ◽  
Author(s):  
Gianluca Bossi ◽  
Giuseppina Mazzaro ◽  
Alessandro Porrello ◽  
Marco Crescenzi ◽  
Silvia Soddu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
P53 Gene ◽  
Wild Type ◽  
Normal Cells ◽  
Tumor Gene Therapy ◽  
Wild Type P53 ◽  
Tumor Gene
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