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Peptide Vector_Biterials Co., Ltd.
Korea Health Industry Development Institute (KHIDI) South Korea flag South Korea
Abstract ID:
Peptide Vector...
Contact Mikyeong Lee
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In the present study, we have constructed a non-viral peptide vector and applied it in the treatment of experimentally induced systemic lupus erythematosus (SLE) like disease in dogs. For therapeutic gene construction, the extracellular domain of canine CTLA-4, and the CH2–CH3 domains of canine immunoglobulin alpha constant region were inserted between the cytomegalovirus promoter and poly-adenylation sequence of bovine growth hormone. The constructed therapeutic gene was ligated to the non-viral synthetic peptide vector and was applied to systemic lupus erythematosus-like disease induced dogs. After gene therapy, clinical signs of systemic lupus erythematosus were reduced dramatically: the anti-nuclear antibody titers and urine protein/creatinine ratios were recovered to normal values, and the skin regained its normal histological features. The peptide vector did not show either tissue specific tropism or host induced immune response.


1. Peptide vector construction


The delivery vector includes leader peptide and linker DNA as shown in Figure 2. Leader peptide has 16 amino acids, which are designed to have functions for membrane fusion and penetration. The linker DNA bridges the leader peptide and a therapeutic gene. The sequences of leader peptide, linker-C, and linker-2 were as follows:


· Leader peptide: Ac-Gly-Leu- Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gly-Arg-Arg-Cys


· Linker-C: 5’-Cys–OO–CTAATACGACTCACTAT-3’ (–OO–: ester bond)


· Linker-2: 3’-GATTATGCTGAGTGAT-5


The N-terminal amino group was replaced with an acetyl group to remove molecular activity. Leader peptide and linker-C were conjugated with a disulfide bond by incubating in an S–S bond buffer (50 mM, Tris, 0.1 mM EDTA, 10 mM DTT, pH 10.5) at 37 °C for 1 h as shown in Step 1. After this procedure, linker-2 was added and incubated at 60 °C for 30 min for the hybridization between linker-C and linker-2 as shown in Step 2. The peptide vector was aliquoted in 100 pmol (20 pmol/mL). The therapeutic gene was amplified by PCR and the product was purified by silica based gel extraction. The purified PCR product (3–5 mg) was ligated into peptide vector (20 pmol) using T4 ligase as shown in Step 3. The final product ligated with the peptide vector was purified by ethanol precipitation and it was rehydrated in phosphate buffered saline and then injected intravenously in two dogs with SLE-like disease.


2. Construction of the therapeutic gene


A therapeutic gene is composed of the extracellular domain of canine CTLA-4 to inhibit the B7:CD28 co-stimulatory pathway and the CH2–CH3 domains of the canine immunoglobulin alpha constant (IGHAC) region to prolong the half-life of therapeutic protein in vivo. The fusion sequence of oncostatin M, CTLA-4 extracellular domain and the CH2–CH3 domains of the IGHAC region was ligated to HindIII and XbaI sites in pcDNA3.1(+) (Invitrogen, USA). Primer pairs were prepared: CMV-F 5’-GCCAGATATACGCGTTGACAT-3’ and BGH-R 5’-GCTTAATGCGCCGCTACA-3’. With these primers, approximately 2213 bp fragments were amplified using pcDNA 3.1(+)/CTLA4Ig as templates.


3. Induction of an SLE-like disease in dogs


We utilized heparan sulfate (HS) to induce an SLE-like disease in eight male dogs. HS is the major glycosaminoglycan of glomerular basement membrane. Autoimmunity to HS has been suggested to be responsible for the induction of tissue damage and kidney dysfunction in SLE in both in vitro and in vivo. All eight dogs developed SLE-like disease. Before the therapeutic gene construction, four of eight SLE induced dogs died. Two of four surviving dogs were treated by peptide vector encoding the therapeutic gene and the other two dogs were used as control.


4. Gene therapy by using Peptide Vector


1) Detection of therapeutic gene


Therapeutic gene was injected intravenously or intraperitoneally into Sprague–Dawley rats (5 weeks old, female). Control rats were injected an equal volume of PBS intravenously. Total RNAs were prepared from the sacrificed rat tissues, which were obtained 3 days after injection by using of Trizol reagent.3) Microscopic observation in gene therapy.


2) Gross morphologic change in gene therapy


In haematoxylin and eosin (H&E) staining, the superficial dermal infiltration of lymphocytes and plasma cells was remarkably reduced as shown in the Fig. 5a (before gene therapy) and 5b (after gene therapy). Hair follicles showed telogen phase before gene therapy (Fig. 5c) and after gene therapy it was recovered to normal anagen phase (Fig. 5d). In immunohistological examination of skin from dogs treated with the therapeutic gene, the deposition of immunoglobulin M (IgM) and C3 (data not shown) along the dermal–epidermal junction of the skins were negligible (Fig. 5f).


Finally, after our gene therapy, clinical signs of systemic lupus erythematosus were reduced dramatically: the anti-nuclear antibody titers and urine protein/creatinine ratios were recovered to normal values, and the skin regained its normal histological features. The peptide vector did not show either tissue specific tropism or host induced immune response.


5.  Safety of Peptide Vector


1) ELISA for detection of anti-peptide vector


IgG antibodies to peptide vector was measured in the sera of control and treated dogs collected on 0, 3, 7, 15, and 30 days after gene therapy as shown in Fig 4. Compared to control, absorbance of anti-peptide vector antibodies in treated dogs was not significantly different. Therefore, it is clear that this method is much safer than virus vector.


 


 

FEATURED
Last Updated Jun 2016
Technology Type THERAPEUTIC
Phase of Development EARLY STAGE
GOVERNMENT INSTITUTE