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Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo.

McNeill E, Iqbal AJ, White GE, Patel J, Greaves DR, Channon KM - Sci Rep (2015)

Bottom Line: We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis.Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells.Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, British Heart Foundation Centre for Research Excellence, John Radcliffe Hospital , University of Oxford, U.K.

ABSTRACT
Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detectable at 5 weeks. We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis. The ability of 35 K-Fc to block more complex pathologies is demonstrated using aortic digests to assess angiotensin II mediated leukocyte recruitment to the aorta. Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells. Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

No MeSH data available.


Related in: MedlinePlus

Expression of 35 K-Fc and 35 K-Fc mutants following hydrodynamic delivery.Mice were injected with 1 or 10 μg 35 K-Fc plasmid. Plasma was harvested 72 hours after injection and immunoprecipitation with protein A/G beads performed. The resulting precipitated proteins were identified by Western blot using anti-Fc and anti-35 K antibodies with purified 35 K-Fc as a positive control and saline injected animals as a negative control (A). Mice received hydrodynamic delivery of 1 μg 35 K-Fc plasmid, individual cohorts of mice were harvested between 2 and 14 days following injection and plasma and liver samples isolated (n = 3–5 per timepoint). Levels of 35 K-Fc in plasma were identified by ELISA (B) and 35 K-Fc message was assessed using specific 35 K taq-man probes in a real-time RT-PCR experiment (C) (*p < 0.05 by one-way ANOVA with post-tests compared to 2-day expression levels). (D) 35 K-Fc message was also assessed in a parallel experiment in liver, spleen and lung to assess the primary site of gene expression. To confirm that 35 K-Fc point mutants were expressed with similar efficiency 1 μg of 35 K-Fc and R89A 35 K-Fc plasmid were injected into mice and plasma harvested for ELISA after 72 hours. No significant difference in the level of plasma protein was detected (E). To determine the biological activity of in vivo expressed R89A 35 K-Fc plasma was added into a real-time chemotaxis assay. A representative trace from the RTCA-DP software showing macrophage migration towards chemoattractant (in the presence of GFP or R89A 35 K-Fc plasma at 1:50 dilution) or buffer control is shown (F). Quantification of the response in the xCELLigence assay showed migration towards CCL5 was significantly inhibited by the presence of R89A 35 K-Fc in the plasma samples (G). (n  =  3 per genotype; T-test p < 0.05 GFP vs R89A 35 K-Fc).
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f2: Expression of 35 K-Fc and 35 K-Fc mutants following hydrodynamic delivery.Mice were injected with 1 or 10 μg 35 K-Fc plasmid. Plasma was harvested 72 hours after injection and immunoprecipitation with protein A/G beads performed. The resulting precipitated proteins were identified by Western blot using anti-Fc and anti-35 K antibodies with purified 35 K-Fc as a positive control and saline injected animals as a negative control (A). Mice received hydrodynamic delivery of 1 μg 35 K-Fc plasmid, individual cohorts of mice were harvested between 2 and 14 days following injection and plasma and liver samples isolated (n = 3–5 per timepoint). Levels of 35 K-Fc in plasma were identified by ELISA (B) and 35 K-Fc message was assessed using specific 35 K taq-man probes in a real-time RT-PCR experiment (C) (*p < 0.05 by one-way ANOVA with post-tests compared to 2-day expression levels). (D) 35 K-Fc message was also assessed in a parallel experiment in liver, spleen and lung to assess the primary site of gene expression. To confirm that 35 K-Fc point mutants were expressed with similar efficiency 1 μg of 35 K-Fc and R89A 35 K-Fc plasmid were injected into mice and plasma harvested for ELISA after 72 hours. No significant difference in the level of plasma protein was detected (E). To determine the biological activity of in vivo expressed R89A 35 K-Fc plasma was added into a real-time chemotaxis assay. A representative trace from the RTCA-DP software showing macrophage migration towards chemoattractant (in the presence of GFP or R89A 35 K-Fc plasma at 1:50 dilution) or buffer control is shown (F). Quantification of the response in the xCELLigence assay showed migration towards CCL5 was significantly inhibited by the presence of R89A 35 K-Fc in the plasma samples (G). (n  =  3 per genotype; T-test p < 0.05 GFP vs R89A 35 K-Fc).

Mentions: As the production of clinical grade, low endotoxin, protein preparations can be time-consuming and expensive we tested whether standard mammalian expression plasmids could be used to express 35 K-Fc directly in vivo. We prepared a low endotoxin plasmid preparation and injected 1 μg or 10 μg by hydrodynamic delivery into C57bl/6J mice and harvested plasma 72 hours later. We detected 35K-Fc in the plasma, which was confirmed to be expressed as the full molecule by Western blotting for both the Fc and 35 K portion of the fusion protein, following immunoprecipitation with Protein A-agarose beads (Fig. 2A). As similar levels of 35 K-Fc were expressed from both doses of plasmid used, we continued our studies using 1 μg of 35 K-Fc plasmid. We confirmed that hydrodynamic delivery of plasmid DNA was well tolerated by the mice by assessing weight following injection, although there was a significant drop in weight this was <2.5% (data not shown). We assessed the pharmacokinetics of 35 K-Fc expression following hydrodynamic delivery. We detected plasma 35 K-Fc at levels in excess of 3 μg/ml for at least 14 days following plasmid injection (Fig. 2B) and saw no significant drop in plasma levels between day 2 and 14. 35 K-Fc was still present in the plasma 5 weeks following injection (mean:734 ng/ml, n = 2), which is in excess of the concentration required for activity of the injected protein.


Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo.

McNeill E, Iqbal AJ, White GE, Patel J, Greaves DR, Channon KM - Sci Rep (2015)

Expression of 35 K-Fc and 35 K-Fc mutants following hydrodynamic delivery.Mice were injected with 1 or 10 μg 35 K-Fc plasmid. Plasma was harvested 72 hours after injection and immunoprecipitation with protein A/G beads performed. The resulting precipitated proteins were identified by Western blot using anti-Fc and anti-35 K antibodies with purified 35 K-Fc as a positive control and saline injected animals as a negative control (A). Mice received hydrodynamic delivery of 1 μg 35 K-Fc plasmid, individual cohorts of mice were harvested between 2 and 14 days following injection and plasma and liver samples isolated (n = 3–5 per timepoint). Levels of 35 K-Fc in plasma were identified by ELISA (B) and 35 K-Fc message was assessed using specific 35 K taq-man probes in a real-time RT-PCR experiment (C) (*p < 0.05 by one-way ANOVA with post-tests compared to 2-day expression levels). (D) 35 K-Fc message was also assessed in a parallel experiment in liver, spleen and lung to assess the primary site of gene expression. To confirm that 35 K-Fc point mutants were expressed with similar efficiency 1 μg of 35 K-Fc and R89A 35 K-Fc plasmid were injected into mice and plasma harvested for ELISA after 72 hours. No significant difference in the level of plasma protein was detected (E). To determine the biological activity of in vivo expressed R89A 35 K-Fc plasma was added into a real-time chemotaxis assay. A representative trace from the RTCA-DP software showing macrophage migration towards chemoattractant (in the presence of GFP or R89A 35 K-Fc plasma at 1:50 dilution) or buffer control is shown (F). Quantification of the response in the xCELLigence assay showed migration towards CCL5 was significantly inhibited by the presence of R89A 35 K-Fc in the plasma samples (G). (n  =  3 per genotype; T-test p < 0.05 GFP vs R89A 35 K-Fc).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Expression of 35 K-Fc and 35 K-Fc mutants following hydrodynamic delivery.Mice were injected with 1 or 10 μg 35 K-Fc plasmid. Plasma was harvested 72 hours after injection and immunoprecipitation with protein A/G beads performed. The resulting precipitated proteins were identified by Western blot using anti-Fc and anti-35 K antibodies with purified 35 K-Fc as a positive control and saline injected animals as a negative control (A). Mice received hydrodynamic delivery of 1 μg 35 K-Fc plasmid, individual cohorts of mice were harvested between 2 and 14 days following injection and plasma and liver samples isolated (n = 3–5 per timepoint). Levels of 35 K-Fc in plasma were identified by ELISA (B) and 35 K-Fc message was assessed using specific 35 K taq-man probes in a real-time RT-PCR experiment (C) (*p < 0.05 by one-way ANOVA with post-tests compared to 2-day expression levels). (D) 35 K-Fc message was also assessed in a parallel experiment in liver, spleen and lung to assess the primary site of gene expression. To confirm that 35 K-Fc point mutants were expressed with similar efficiency 1 μg of 35 K-Fc and R89A 35 K-Fc plasmid were injected into mice and plasma harvested for ELISA after 72 hours. No significant difference in the level of plasma protein was detected (E). To determine the biological activity of in vivo expressed R89A 35 K-Fc plasma was added into a real-time chemotaxis assay. A representative trace from the RTCA-DP software showing macrophage migration towards chemoattractant (in the presence of GFP or R89A 35 K-Fc plasma at 1:50 dilution) or buffer control is shown (F). Quantification of the response in the xCELLigence assay showed migration towards CCL5 was significantly inhibited by the presence of R89A 35 K-Fc in the plasma samples (G). (n  =  3 per genotype; T-test p < 0.05 GFP vs R89A 35 K-Fc).
Mentions: As the production of clinical grade, low endotoxin, protein preparations can be time-consuming and expensive we tested whether standard mammalian expression plasmids could be used to express 35 K-Fc directly in vivo. We prepared a low endotoxin plasmid preparation and injected 1 μg or 10 μg by hydrodynamic delivery into C57bl/6J mice and harvested plasma 72 hours later. We detected 35K-Fc in the plasma, which was confirmed to be expressed as the full molecule by Western blotting for both the Fc and 35 K portion of the fusion protein, following immunoprecipitation with Protein A-agarose beads (Fig. 2A). As similar levels of 35 K-Fc were expressed from both doses of plasmid used, we continued our studies using 1 μg of 35 K-Fc plasmid. We confirmed that hydrodynamic delivery of plasmid DNA was well tolerated by the mice by assessing weight following injection, although there was a significant drop in weight this was <2.5% (data not shown). We assessed the pharmacokinetics of 35 K-Fc expression following hydrodynamic delivery. We detected plasma 35 K-Fc at levels in excess of 3 μg/ml for at least 14 days following plasmid injection (Fig. 2B) and saw no significant drop in plasma levels between day 2 and 14. 35 K-Fc was still present in the plasma 5 weeks following injection (mean:734 ng/ml, n = 2), which is in excess of the concentration required for activity of the injected protein.

Bottom Line: We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis.Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells.Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, British Heart Foundation Centre for Research Excellence, John Radcliffe Hospital , University of Oxford, U.K.

ABSTRACT
Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detectable at 5 weeks. We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis. The ability of 35 K-Fc to block more complex pathologies is demonstrated using aortic digests to assess angiotensin II mediated leukocyte recruitment to the aorta. Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells. Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

No MeSH data available.


Related in: MedlinePlus