Systemic circulation of poly(l-lysine)/DNA vectors is influenced by polycation molecular weight and type of DNA: differential circulation in mice and rats and the implications for human gene therapy

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2221-2229 ◽  
Author(s):  
Christopher M. Ward ◽  
Martin L. Read ◽  
Leonard W. Seymour
Keyword(s):  
Gene Therapy ◽  
Molecular Weight ◽  
Kupffer Cells ◽  
Physiological Saline ◽  
Systemic Circulation ◽  
Complement C3 ◽  
Systemic Delivery ◽  
Dna Complexes

Abstract Effective gene therapy for diseases of the circulation requires vectors capable of systemic delivery. The molecular weight of poly(l-lysine) (pLL) has a significant effect on the circulation of pLL/DNA complexes in mice, with pLL211/DNA complexes displaying up to 20 times greater levels in the blood after 30 minutes compared with pLL20/DNA. It is shown that pLL20/DNA complexes fix mouse complement C3 in vitro, independent of immunoglobulin binding; are less soluble in the blood in vivo; bind erythrocytes; are rapidly removed by the liver, where they associate predominantly with Kupffer cells; and result in a rapid increase in hepatic leukocytes expressing high levels of complement receptor 3 (CR3). The circulation properties of these complexes are also dependent on the type of DNA used, with circular plasmid DNA complexes exhibiting increased circulation compared with linear DNA. PLL211/DNA complexes bind erythrocytes and associate with Kupffer cells but, in contrast, do not fix mouse complement in vitro and are unaffected by the type of DNA used. In rats, both types of complexes produce hematuria and are rapidly removed from the circulation. Correlation of in vivo and in vitro results suggests that the solubility of complexes in physiological saline and species-matched complement fixation and erythrocyte lysis may correlate with systemic circulation. Analysis using human blood in vitro shows no hemolysis, but both types of complexes fix complement and bind IgG, suggesting that pLL/DNA complexes may be rapidly cleared from the human circulation.

scholarly journals Immunity against both polyomavirus VP1 and a transgene product induced following intranasal delivery of VP1 pseudocapsid–DNA complexes

2001 ◽  
Vol 82 (11) ◽  
pp. 2791-2797 ◽  
Author(s):  
B. Clark ◽  
W. Caparrós-Wanderley ◽  
G. Musselwhite ◽  
M. Kotecha ◽  
B. E. Griffin
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Dna Complexes ◽  
Cell Monolayers ◽  
Molar Ratios ◽  
Repeated Use ◽  
Murine Polyomavirus ◽  
Delivery Vehicles

Murine polyomavirus VP1 virus-like particles (VLPs) can bind plasmid DNA and transport it into cells both in vitro and in vivo. Long-term expression of the transgene can be observed, suggesting that VP1 VLPs may be used as DNA delivery vehicles for gene therapy. In this study we have analysed the in vitro efficiency of transfection using different DNA/VLP molar ratios and the immune response induced following intranasal administration of these complexes to mice. Our results indicate that in short-term in vitro culture VP1 VLP–DNA complexes appear to be as efficient as DNA alone at transfecting cell monolayers. They also show that VP1 VLPs are very immunogenic, inducing high proliferative cell responses and both serum and mucosal antibodies. Moreover, VP1 VLP–DNA complexes appear to be capable of inducing a stronger immune response to the transgene product (β-galactosidase) than immunization with DNA only. The results suggest that polyomavirus VP1 VLPs derived from the wild-type sequence may be too immunogenic for repeated use as gene delivery vehicles in gene therapy. However, due to their high immunogenicity and apparent adjuvant properties, they could be modified and used as vaccines either on their own or complexed with DNA.

scholarly journals Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages

2008 ◽  
Vol 89 (5) ◽  
pp. 1097-1105 ◽  
Author(s):  
Hidde J. Haisma ◽  
Jan A. A. M. Kamps ◽  
Gera K. Kamps ◽  
Josee A. Plantinga ◽  
Marianne G. Rots ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Kupffer Cells ◽  
Transgene Expression ◽  
Liver Toxicity ◽  
Viral Vector ◽  
Adenovirus Vector ◽  
Liver Macrophages ◽  
Polyinosinic Acid

Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.

A Single Intramuscular Injection of AAV-8 Vector Expressing MDA7/IL-24 Efficiently Suppresses Tumor Growth Mediated by Multiple Anti-Cancer Mechanisms In Lymphoma Model Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1474-1474
Author(s):  
Nanya Wang ◽  
Bai Fan ◽  
Noriko Miyake ◽  
Koichi Miyake ◽  
Takashi Shimada
Keyword(s):  
Gene Therapy ◽  
Tumor Growth ◽  
Tumor Cell ◽  
Murine Model ◽  
Conflicts Of Interest ◽  
Quadriceps Muscle ◽  
C2c12 Cells ◽  
Systemic Delivery

Abstract Abstract 1474 Melanoma differentiation-associated gene-7/interleukin-24 (MDA7/IL-24) selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and anti-angiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for use in a new anticancer gene therapy. To examine the feasibility of adeno-associated virus (AAV) vectors expressing MDA7/IL-24 in systemic cancer gene therapy for lymphoma, we generated an AAV type 8 vector expressing MDA7/IL-24 (AAV-IL24). In vitro studies showed that medium conditioned by AAV-IL24-transduced C2C12 cells induces tumor cell-specific apoptosis against murine lymphoma cell line (A20 cell). To assess the in vivo effects of muscle targeted AAV-mediated systemic delivery of MDA7/IL-24 we established a lymphoma murine model in which the A20 cells expressing luciferase gene was inoculated into the caudal vein of BALB/C. Using this lymphoma murine model, we can detect the tumor growth and metastases by a real-time in vivo imaging analyze system (IVIS). After single injection of AAV-IL24 (1.5×1011 vg/body) into the right quadriceps muscle of the lymphoma model mice, tumor cell growth was monitored by IVIS. ELISA analysis showed high level of IL-24 was detected in plasma of treated mice (263±16 ng/ml). Suppression of tumor growth was observed in AAV-IL24 injected mice compared to control GFP expressing AAV injected mice (1.3×108vs.2.6×108 photon/sec; p<0.05). Survival effect was also detected in AAV-IL24 mice (66±6 vs. 47±5 days; p<0.03). In addition, TUNEL analyses showed significant induction of tumor cell-specific apoptosis within the tumors and suppression of angiogenesis was also detected in AAV-IL24 treated mice. Finally, immune modulating activity of induction of Th1 cytokines (IL-6, TNFα, IFNγ) secretion was observed after AAV-IL24 injection. These results clearly demonstrate that continuous systemic delivery of MDA7/IL-24 can serve as an effective treatment for lymphoma. Thus, AAV type 8 vector-mediated systemic deliverY of MDA7/IL-24 represents a potentially promising new approach to treat lymphoma. Disclosures: No relevant conflicts of interest to declare.

Effects of Unfractionated and Low Molecular Weight Heparins on Platelet Thromboxane Biosynthesis “In Vivo”

1994 ◽  
Vol 72 (06) ◽  
pp. 942-946 ◽  
Author(s):  
Raffaele Landolfi ◽  
Erica De Candia ◽  
Bianca Rocca ◽  
Giovanni Ciabattoni ◽  
Armando Antinori ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Low Molecular Weight Heparin ◽  
Primary Source ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
Heparin Administration ◽  
To Receive

SummarySeveral “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.

Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

1964 ◽  
Vol 12 (01) ◽  
pp. 232-261 ◽  
Author(s):  
S Sasaki ◽  
T Takemoto ◽  
S Oka
Keyword(s):  
Molecular Weight ◽  
Dextran Sulfate ◽  
Anticoagulant Activity ◽  
Molecular Structures ◽  
Severe Reaction ◽  
Clinical Use ◽  
Molecular Weights ◽  
Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

Neutralization of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin by Protamine Sulfate in Rats

1986 ◽  
Vol 56 (03) ◽  
pp. 318-322 ◽  
Author(s):  
V Diness ◽  
P B Østergaard
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Thrombus Formation ◽  
Experimental Models ◽  
Protamine Sulfate ◽  
Low Molecular Weight ◽  
Antithrombotic Effect ◽  
Higher Doses

SummaryThe neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized.In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.

Studies on Metabolism of Urokinase and Mechanism of Thrombolysis by Urokinase

1979 ◽  
Vol 42 (03) ◽  
pp. 885-894 ◽  
Author(s):  
Tatsuo Ueno ◽  
Norio Kobayashi ◽  
Tadashi Maekawa
Keyword(s):  
Molecular Weight ◽  
Plasma Clearance ◽  
Radioactive Material ◽  
Optimal Dosage ◽  
Plasma Clot ◽  
Optimal Dosage Regimen ◽  
Arterial Thrombus ◽  
Contact Experiment

SummaryPharmacokinetics of intravenously injected 125I-labeled urokinase (125I-UK) of a molecular weight of 33,000 daltons in normal rabbits and patients with various diseases were investigated. The plasma clearance of 125I-UK in rabbits was described by a biexponential curve within six hours with a half-life of 8 minutes, 2.3 hours, respectively. The radioactivity in the liver and kidneys 15 minutes after iv injection with 125I-UK was 9.6% and 14.0% of the radioactivity injected, respectively. Approximately 80% of the total radioactive material injected was excreted in the urine in 18 hours. No increase in activator activity in the urine was observed after a large amount of UK injection. Activity uptake of 125I-UK by experimentally induced arterial thrombus was little. Lysis of the stasis thrombus was produced by injecting 7.5 × 104 IU of UK in only one out of 8 rabbits. In vitro contact experiment revealed that transfer of 125I-UK to plasma clot is slow (24 hours for 10% of 125I-UK by plasma clot). In 4 patients plasma clearance of 125I-UK was essentially similar to that in rabbits. From the results obtained optimal dosage regimen of UK administration for complete thrombolysis in vivo was discussed.

SiRNA technology, the gene therapy of the future?

2008 ◽  
Vol 149 (4) ◽  
pp. 153-159 ◽  
Author(s):  
Zsuzsanna Rácz ◽  
Péter Hamar
Keyword(s):  
Gene Therapy ◽  
Short Interfering Rna ◽  
The Future ◽  
Interfering Rna

A genetikában új korszak kezdődött 17 éve, amikor a petúniában felfedezték a koszuppressziót. Később a koszuppressziót azonosították a növényekben és alacsonyabb rendű eukariótákban megfigyelt RNS-interferenciával (RNSi). Bár a növényekben ez ősi vírusellenes gazdaszervezeti védekezőmechanizmus, emlősökben az RNSi élettani szerepe még nincs teljesen tisztázva. Az RNSi-t rövid kettős szálú interferáló RNS-ek (short interfering RNA, siRNS) irányítják. A jelen cikkben összefoglaljuk az RNSi történetét és mechanizmusát, az siRNS-ek szerkezete és hatékonysága közötti összefüggéseket, a célsejtbe való bejuttatás virális és nem virális módjait. Az siRNS-ek klinikai alkalmazásának legfontosabb akadálya az in vivo alkalmazás. Bár a hidrodinamikus kezelés állatokban hatékony, embereknél nem alkalmazható. Lehetőséget jelent viszont a szervspecifikus katéterezés. A szintetizált siRNS-ek ismert mellékhatásait szintén tárgyaljuk. Bár a génterápia ezen új területén számos problémával kell szembenézni, a sikeres in vitro és in vivo kísérletek reményt jelentenek emberi betegségek siRNS-sel történő kezelésére.

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

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