Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Nov:70:84-93.
doi: 10.1016/j.biomaterials.2015.08.004. Epub 2015 Aug 5.

Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B

Jin Su  1 , Liqing Zhu  2 , Alexandra Sherman  2 , Xiaomei Wang  2 , Shina Lin  1 , Aditya Kamesh  1 , Joey H Norikane  3 , Stephen J Streatfield  3 , Roland W Herzog  4 , Henry Daniell  5
Affiliations

Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B

Jin Su et al. Biomaterials. 2015 Nov.

Abstract

Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ∼2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP(+) regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ∼870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft(2) per annum yielding 24,000-36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs.

Keywords: Chloroplast; Hemophilia; Lettuce; Molecular pharming; Oral tolerance; cGMP plant production.

PubMed Disclaimer

Conflict of interest statement

Competing financial interests

Henry Daniell is an inventor in several US and international patents on chloroplast transformation technology to produce biopharmaceuticals, in particular induction of oral tolerance. Henry Daniell and Roland Herzog are co-inventors in hemophilia patents. Novo Nordisk is currently funding the hemophilia project.

Figures

Figure 1
Figure 1. CTB-FIX lettuce chloroplast expression vector and evaluation of site-specific integration the lettuce chloroplast genome
(A) Schematic diagram of CTB-FIX lettuce expression vector pLS-CTB-FIX. Homologous chloroplast genome flanking sequences include 16S (16S rRNA), isoleucine tRNA (trnI), alanine tRNA (trnA) genes. A glycine-proline-glycine-proline hinge and furin cleavage site (RRKR) are inserted between CTB and the FIX sequence. The CTB-FIX expression is regulated by the lettuce psbA promoter-5′ UTR (PpsbA) and 3′ UTR (TpsbA). The selection marker gene aadA (encoding aminoglycoside 3′-adenylyltransferase gene to confer spectinomycin resistance) is driven by a ribosomal RNA operon promoter (Prrn) with GGAG ribosome binding site. (B) PCR analysis of the CTB-FIX transplastomic lines. The primer annealing sites (16S-Fw/3M, 5P/2M and CTB-Fw/FIX-Rv1) for PCR analysis of control and transplastomic lines are shown in Figure 1a. L1, L2 and L3 represent three independent transplastomic lines. (C) Evaluation of homoplasmy in CTB-FIX-transplastomic lines. Total lettuce DNA was digested with HindIII and probed with 32P-labeled 1.12 kb trnI/trnA flanking region fragment. Untransformed line generates a 9.1 kb hybridizing fragment while transplastomic lines generate 12.6 kb fragment due to site specific integration of the transgene cassette.
Figure 2
Figure 2. Evaluation of CTB-FIX expression in the lettuce transplastomic lines
(A) Western blot analysis of CTB-FIX in three independent lines and WT controls. Plant total leaf proteins (5 μg/lane) were loaded from WT and CTB-FIX plants. CTB, 3 ng per lane. L1, L2, L3: 3 independent lettuce transplastomic lines. WT, untransformed wild type plant. Antibody titers: rabbit anti-CTB polyclonal antibody 1:10,000; goat anti-FIX polyclonal antibody 1:2,000. (B) Quantitation of CTB-FIX expression at different developmental stages. Total leaf protein (1.5 μg per lane) was loaded from young, mature and old leaves (2.5-months-old plants grown in the greenhouse). Probe: anti-CTB rabbit polyclonal antibody (titer 1:10,000). Right bar graph shows CTB-FIX concentrations of young, mature and old leaves. Data shown are means ± SD of two replicates from leaves collected from two independent lines. (C) Non-reducing gel western blot analysis. Probe, anti-CTB rabbit polyclonal antibody (titer 1:10,000). Protein concentration loaded: CTB, 3 ng per lane; CTB-FIX (L1, L2 and L3) and WT leaf extracts, 5 μg (TLP) per lane.
Figure 3
Figure 3. Evaluation of CTB-FIX concentration and stability after long term storage of lyophilized lettuce leaves
(A) Comparison of CTB-FIX protein concentration of lyophilized CTB-FIX lettuce leaves with the frozen leaf samples. 1× 40× indicate fold of dilution. One fold (1×) equals 200 μg (in3 weight) of leaf ground powder. (B) Evaluation of CTB-FIX protein stability in lyophilized lettuce leaves during long term storage at ambient temperature. Two independent blots by loading either 2.0 μg or 1.5 μg of TLP per lane are shown. Probe: anti-CTB rabbit polyclonal antibody (titer, 1:10,000). Right bar graph shows the CTB-FIX concentrations of the 4 batches of lyophilized leaves after different periods of storage (2, 6, 12 and 24 months). Data shown are means ± SD of three independent experiments. (C) GM1 binding assay. CTB: CTB standard protein (5 ng). Lyo-2M, Lyo-6M, Lyo-12M, Lyo-24M: Total soluble protein (TSP, 100 μg) from the lyophilized CTB-FIX expressing leaves after storage for 2, 6, 12 and 24 months at room temperature. Lyo-WT, protein extract (100 μg, TSP) from lyophilized and untransformed wild type leaves. BSA, bovine serum albumin (100 μg). Data shown are means ± SD of two independent experiments. (D) Comparative analysis of CTB-FIX protein concentrations in lyophilized lettuce leaves from Fraunhofer (Fraun, hydroponic system) with leaf samples collected from the Daniell lab greenhouse at University of Pennsylvania (UPenn, potted soils). 1.5 μg TLP per lane was loaded. Probe: anti-CTB rabbit polyclonal antibody (titer, 1:10,000). Left panel represents results of the 1st leaf harvest (plant ages: Fraunhofer, 32-days-old; UPenn, 66-days-old) and the right panel shows results of the 2nd leaf harvest (plant ages: Fraunhofer, 51-days-old; UPenn, 86-days-old).
Figure 4
Figure 4. Industrial production of FIX bioencapsulated in lettuce cells
(A) Biomass production of CTB-FIX lettuce. CTB-FIX lettuce plants were grown in potted soils in the Daniell lab greenhouse at the University of Pennsylvania or in Fraunhofer cGMP hydroponic system (Upper 2 panels). The lower 2 panels show the growth status of CTB-FIX lettuce (cv. Simpson Elite, 24 days old) and wild type (WT) lettuce (cv. Simpson Elite, 24 days old). (B) Capsule preparation. A large scale production of FIX-expressing lettuce was established in Fraunhofer USA (Newark, DE). After harvesting and lyophilization (Lyophilizer: Genesis 35×L, SP Scientific, Stone Ridge, NY) of the fresh leaves, the freeze-dried FIX leaves were powdered and prepared as capsules.
Figure 5
Figure 5. FIX antigen delivery to the small intestine of hemophilia B mice
(A) FIX (red) delivered to epithelium of intestinal villi and to Peyer’s patches (PP). DAPI was used to stain nuclei blue. (B) Examples of co-localization (arrows) of FIX antigen and CD11c+ cells, indicating antigen delivery to DC in PP. (C) Examples of co-localization (arrows) of FIX antigen and CD11c+ cells, indicating antigen delivery to DC in villi. Mice had received oral gavages of CTB-FIX expressing lettuce (but no i.v. FIX). (D) No FIX stain was observed in mice that received oral gavage of WT lettuce (shown), or in intestinal sections of CTB-FIX fed mice when the primary antibody against FIX was omitted during staining (not shown). Original magnification for all panels: 200×. (E) Systemic human FIX levels as measured by ELISA 2 and 5 hrs after oral gavage of 10 μg CTB-FIX bioencapsulated in lettuce or tobacco cells. Data shown are average ± SD (n=3/group). Differences between lettuce and tobacco were not significant (“ns”).
Figure 6
Figure 6. Suppression of inhibitor and IgE formation against FIX in hemophilia B mice
(A) Mice were given two gavages weekly (of different CTB-FIX doses) of lettuce cells and were challenged with i.v. injections of FIX. (B) Anti-FIX titers 1 week after the last i.v. injection of FIX are graphed as inhibitor titers in BU/ml. (C) IgG1, 2a, and 2b anti-FIX titers. (D) IgE anti-FIX titers. (E) Frequencies of LAP+CD25−CD4+ Treg in the spleen, mesenteric lymph nodes, Peyer’s patches, and control (inguinal) lymph nodes of FIX fed and control mice. Flow plots on the left show individual representative examples. Graph to the right shows results for individual mice (n=5/group) as well as averages ± SD.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Walsh G. Biopharmaceutical benchmarks. Nat Biotechnol. 2014;32:992–1000. - PubMed
    1. Jin SX, Daniell H. Engineered chloroplast genome just got smarter. Trends Plant Sci. 2015 in press. - PMC - PubMed
    1. Chan HT, Daniell H. Plant-made vaccines against human diseases–are we there yet? Plant Biotechnol J. 2015 in press. - PMC - PubMed
    1. Kwon KC, Verma D, Singh ND, Herzog R, Daniell H. Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells. Adv Drug Deliv Rev. 2013;65:782–99. - PMC - PubMed
    1. Kwon KC, Nityanandam R, New JS, Daniell H. Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells. Plant Biotechnol J. 2013;11:77–86. - PMC - PubMed

Publication types

MeSH terms