scholarly journals Adenovirus-Mediated Suicide SCLC Gene Therapy Using the Increased Activity of the hTERT Promoter by the MMRE and SV40 Enhancer

2005 ◽  
Vol 69 (1) ◽  
pp. 56-62 ◽  
Author(s):  
Joon-Seok SONG
Keyword(s):  
Gene Therapy ◽  
Htert Promoter ◽  
Sv40 Enhancer ◽  
Increased Activity

scholarly journals Modification of the hTERT promoter by heat shock elements enhances the efficiency and specificity of cancer targeted gene therapy

2016 ◽  
Vol 32 (3) ◽  
pp. 244-253 ◽  
Author(s):  
Xiaolong Wang ◽  
PeiHua Zhou ◽  
XueJun Sun ◽  
GuangBing Wei ◽  
Li Zhang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Heat Shock ◽  
Htert Promoter ◽  
Targeted Gene Therapy

A Novel Hybrid Promoter ARE-hTERT for Cancer Gene Therapy

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 66-73
Author(s):  
S. V. Kalinichenko ◽  
M. V. Shepelev ◽  
P. N. Vikhreva ◽  
I. V. Korobko
Keyword(s):  
Gene Therapy ◽  
Cancer Cells ◽  
Cytosine Deaminase ◽  
Chimeric Protein ◽  
Gene Promoter ◽  
Cancer Gene Therapy ◽  
Cancer Gene ◽  
Htert Promoter ◽  
Hybrid Promoter

describe a novel hybrid tumor-specific promoter, ARE-hTERT, composed of the human TERT gene promoter (hTERT) and the antioxidant response element (ARE) from the human GCLM gene promoter. The hybrid promoter retains the tumor specificity of the basal hTERT promoter but is characterized by an enhanced transcriptional activity in cancer cells with abnormal activation of the Nrf2 transcription factor and upon induction of oxidative stress. In the in vitro enzyme-prodrug cancer gene therapy scheme, ARE-hTERT promoter-driven expression of CD : UPRT (yeast cytosine deaminase : uracil phosphoribosyltransferase) chimeric protein induced a more pronounced death of cancer cells either upon treatment with 5-fluorouracil (5FC) alone or when 5FC was combined with chemotherapeutic drugs as compared to the hTERT promoter. The developed hybrid promoter can be considered a better alternative to the hTERT promoter in cancer gene therapy schemes.

scholarly journals A novel cancer vaccine strategy with combined IL-18 and HSV-TK gene therapy driven by the hTERT promoter in a murine colorectal cancer model

2014 ◽  
Vol 45 (4) ◽  
pp. 1412-1420 ◽  
Author(s):  
KOSUKE HIGASHI ◽  
SHOICHI HAZAMA ◽  
ATSUHIRO ARAKI ◽  
KIYOSHI YOSHIMURA ◽  
NORIO IIZUKA ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Gene Therapy ◽  
Cancer Vaccine ◽  
Htert Promoter ◽  
Cancer Model ◽  
Vaccine Strategy

Unexpected Role of PACE/Furin Cleavage Site in FVIII Biology: Implications for Hemophilia a Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 105-105
Author(s):  
Joshua I Siner ◽  
Julie M Crudele ◽  
Courtney T Connolly ◽  
Shangzhen Zhou ◽  
Elizabeth P. Merricks ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Amino Acid ◽  
Cleavage Site ◽  
Manufacturing Systems ◽  
Hemophilia A ◽  
Wild Type ◽  
Furin Cleavage ◽  
Intracellular Processing ◽  
Liver Gene ◽  
Increased Activity

Abstract The paired basic amino acid cleaving enzyme (PACE)/Furin is a protein convertase system that plays a vital role in several biological processes, including coagulation. The propeptide processing of human FIX by PACE/Furin is a critical posttranslational modification, so cells co-expressing PACE/Furin and FIX are used for production of clinical recombinant protein. In the development of recombinant B-domain deleted (BDD) FVIII for hemophilia A (HA), a 14 amino acid B-domain sequence containing a putative cleavage site for PACE/Furin was retained because it was believed to be critical for intracellular processing and secretion. In contrast to FIX, we report here a surprising detrimental effect of PACE/Furin in FVIII activity and intracellular processing and secretion. We engineered a human FVIII variant where the PACE/Furin site at residues 1645-1648 was deleted from FVIII-BDD (FVIII-ΔP/F). Notably, FVIII-ΔP/F exhibits a 3-fold increased activity over FVIII-BDD (p=0.0004) in a 2-stage APTT assay. Moreover, the A2-domain dissociation of activated FVIII-ΔP/F was also 3-fold longer compared to FVIII-BDD, suggesting a more stable activated FVIII molecule. The amount of FVIII secreted from stably transduced BHK cells was about 3-fold higher for FVIII-ΔP/F than for FVIII-BDD. Conversely, the amount of intracellular FVIII antigen was lower for FVIII-ΔP/F than for FVIII-BDD. To confirm that PACE/Furin was implicated in the underlying mechanisms for the observed differences in FVIII secretion, we inhibited PACE/Furin in FVIII-BDD producing BHKs by transducing them with vectors expressing an engineered α1-antitrypsin variant (haat-PDX) that specifically inhibits PACE/Furin. This resulted in a 40% increase in FVIII secretion (p=0.017) and a decrease in intracellular FVIII-BDD, whereas transduction with the haat-wild type control, which does not inhibit PACE/Furin, did not significantly change the amount of FVIII secreted (p=0.32). Importantly, the secretion and intracellular levels of FVIII-ΔP/F were not affected by the inhibition of PACE/Furin by haat-PDX, indicating that the secretion of this FVIII variant does not benefit from further inhibition of PACE/Furin cleavage. Together these data suggest that the increased secretion of FVIII-ΔP/F compared to FVIII-BDD is due to the former circumventing PACE/Furin. Furin is ubiquitously expressed in mammal tissues. In a stringent cellular model, we used LoVo, a unique human cell line that lacks functional Furin to determine whether expression of FVIII-ΔP/F and FVIII-BDD would differ, as we have observed in cells expressing Furin. Interestingly, the secretion of FVIII-ΔP/F and FVIII-BDD were comparable. This result confirms that FVIII-BDD is secreted better in the absence of Furin. In summary, our novel variant FVIII-ΔP/F exhibits enhanced secretion primarily by bypassing PACE/Furin cleavage; inhibiting this cellular process also enhances the secretion of FVIII. Futhermore in vivo experiments also demonstrated a beneficial effect of FVIII-ΔP/F: HA mice (n=4-7/dose) given adeno-associated viral 8 (AAV8) vectors for liver gene expression of FVIII-ΔP/F resulted in a 3-fold higher circulating FVIII levels than FVIII-BDD-expressing mice (p=0.025). These exciting results from human FVIII-ΔP/F prompt us to test this variant HA canine model. First we found that recombinant canine FVIII with the entire PACE/Furin site deleted (cFVIII-ΔP/F) had increased activity in a 2-stage aPTT assay compared to wild-type cFVIII-BDD. Injection of cFVIII-ΔP/F effectively corrects the hemophilia coagulopathy in two HA dogs. Further, AAV8 liver gene therapy with cFVIII-ΔP/F in additional two HA dogs at doses of ~6 x 1012 vg/kg, a log lower than previously used for canine FVIII-BDD AAV8 gene therapy, resulted in therapeutic levels of cFVIII and shortening of clotting times. Preliminary data on injection of cFVIII-BDD protein was well tolerated in cFVIII-ΔP/F-expressing dogs. In conclusion, these data suggest that PACE/Furin cleavage of FVIII hampers protein biological activity. FVIII variants lacking PACE/Furin recognition sequences are secreted more efficiently and exhibit improved hemostatic effects in both protein- and gene-based strategies. Inhibition of PACE/Furin in manufacturing systems for recombinant human FVIII may increase the yields of protein production. Thus these strategies have a strong rationale for translation to HA therapy. Disclosures No relevant conflicts of interest to declare.

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