Producing Human Therapeutic Proteins in Plastids

Plastid transformation technology is set to become a major player in the production of human therapeutic proteins. Protein expression levels that can be achieved in plant plastids are hundreds of times greater than the expression levels generally obtained via nuclear transformation. Plastids can produce human proteins that are properly folded and are biologically active. Effective protein purification strategies and strategies that can achieve inducible plastid gene expression are being developed within the system. Plastid transformation technology has been extended to edible plant species, which could minimize down-stream processing costs and raises the possibility of “edible protein therapies”. The system is limited by the fact that plastid-produced proteins are not glycosylated and that, at the moment, it can be difficult to predict protein stability within the plastid. The high level of protein expression that can be obtained in plastids could make it possible to produce high-value therapeutic proteins in plants on a scale that could be accommodated in contained glasshouse facilities and still be economically viable. Growing plastid-transformed plants under contained conditions, and coupled with the level of bio-safety conferred by maternal inheritance of plastid transgenes, would address many of the social and environmental concerns relating to plant based production of human therapeutic proteins

WTA immunization reduces MW2 CA-MRSA infection in WT mice while no difference is seen in the absence of MBL.

<p>(2A) Abscess formation. Mice immunized with PBS control or WTA were infected 20 days after the last immunization. Abscess formation was examined on day 10 following systemic infection with MW2 CA-MRSA as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069739#s2" target="_blank">Materials and Methods</a>. Abscess formation is expressed as numbers of mice with abscess and total mice in each group. * indicates p<0.0001 against all other groups (Likelihood Ratio). (2B) Bacterial load in the kidney. Bacterial titers were measured in homogenates of two combined kidneys and are expressed as cfu/g of kidneys in a box plot. Numbers of mice used are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069739#pone-0069739-g002" target="_blank">figure 2A</a>. * and ** indicates p<0.05 and p<0.001, respectively compared to WT immunized with PBS control (Nonparametric comparisons for each pair by Wilcoxon methods).</p

Abscess formation in systemic MRSA infection.

*<p>indicates p<0.0001 (Likelihood ratio) compared to WT in MW2 CA-MRSA infection.</p

Serum from WTA-immunized mice inhibits bacterial growth in whole blood assays <i>ex vivo</i>.

<p>Two MRSA strains, COL HA-MRSA (4A and 4B) and MW2 CA-MRSA, (4C and 4D) were used. Bacteria were incubated with whole blood from MBL KO (4A and 4C) or WT mice (4B and 4D) with serum from MBL KO mice immunized with either PBS control or WTA, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069739#s2" target="_blank">Materials and Methods</a>. Bacterial titers are expressed as mean <b>±</b> SD in each group of four samples. Each sample was measured in duplicate and the average measurement was used for statistical analysis. Representative results from two repeated experiments are shown. *, **, and *** indicate p<0.05, 0.001, and 0.0001 (Student's t-test), respectively.</p

Enhanced cardiac pathology and fibrosis in MBL^−/− infected with <i>T.</i><i>cruzi</i> Colombiana strain.

<p>WT and MBL^−/− mice were infected with trypomastigotes of the Colombiana strain of <i>T. cruzi</i> and hearts from these animals obtained 5 weeks after infection. Hearts were formalin-fixed, paraffin embedded, stained with H&E or Gomori’s trichrome and scored for pathology and fibrosis, respectively, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047835#s4" target="_blank">Materials and Methods</a> (A). Right panels show micrographs of Gomori’s trichrome staining for each group. Bars indicate the standard error of the mean (SEM). Uninfected, or WT and MBL^−/− mice infected as in (A) were harvested 5 wks later for determination of hydroxyproline content in total heart (B). Each symbol corresponds to an individual animal, dashed lines represent the mean of each group and solid lines the standard error of the mean (SEM). Heart samples from uninfected controls or from WT and MBL^−/− mice infected as in (A) were obtained and mRNA accumulation of Collagen-1, -3 and -6 determined by real-time PCR (C). The experiments shown are representative of two performed. *, Indicates statistically significant differences between infected WT and MBL^−/− groups.</p

<i>T. cruzi</i> infection induces MBL expression <i>in vivo</i>.

<p>WT and MyD88^−/− mice were infected with trypomastigotes of the Y strain of <i>T. cruzi</i> and 9 days after infection, MBL-A and MBL-C mRNA accumulation was determined in spleens from infected and uninfected animals by real-time PCR (A). Detection of MBL-C protein was also performed by immunofluorescence microscopy in splenic sections obtained from WT mice infected as above (B). Micrograph shows MBL-C (red) and B220 (blue) staining in both the red (RP) and white pulp (WP). Scale bar, 100 µm. Infected MBL^−/− spleens were included as a control. All panels shown are representative of 2 independent experiments using 3 to 4 mice per group. Bars indicate the standard error of the mean (SEM). Sera from patients with acute, chronic or indeterminate Chagas’ disease were assayed for MBL as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047835#s4" target="_blank">Material and Methods</a> (C). *, Indicates statistically significant differences between acute vs indeterminate Chagas’ disease groups.</p

Comparison of <i>ΔΨ</i>_<i>T</i> in 17 "bull’s eye" segments Results shown for five control (blue squares) and three pigs with injury to the LAD territory (red circles).

<p>Comparison of <i>ΔΨ</i>_<i>T</i> in 17 "bull’s eye" segments Results shown for five control (blue squares) and three pigs with injury to the LAD territory (red circles).</p

Parametric images of V_T and ΔΨ_T.

<p>Each panel of three x two images shows short axis, vertical and horizontal slices. Images of a representative control pig are shown in the left panel. Images of a representative scar pig are shown in the right panel. The top row of each panel depicts the TPP⁺ volume of distribution and bottom row the membrane potential.</p

Typical ¹⁸F-TPP⁺ SUV image, integrated from 60–120 minutes after IV bolus injection.

<p>SUV is highest in liver, followed by LV myocardium, with lower activity in the visible in bone marrow.</p

Volume of distribution model for¹⁸F-TPP⁺ in a PET image voxel.

<p>The outer black line represents the voxel boundary. C_p, C_inter, C_cyto, and C_mito represent the concentrations of the plasma, interstitial space, cytosol, and mitochondria respectively. The arrows represent ¹⁸F-TPP⁺ transport between the different compartments. <i>f</i>_ECS represents the voxel volume fraction occupied by ECS and <i>f</i>_mito represents the cellular volume fraction occupied by mitochondria.</p