scholarly journals Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2474
Author(s):  
Kian Chuan Sia ◽  
Zhen Ying Fu ◽  
Roy Y. Calne ◽  
Amit C. Nathwani ◽  
Kok Onn Lee ◽  
...  
Keyword(s):  
Human Insulin ◽  
Liver Toxicity ◽  
Insulin Gene ◽  
Diabetic Mice ◽  
Insulin Expression ◽  
Human Insulin Gene ◽  
Vector Genome ◽  
Virus Serotype

We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3′ albumin enhancer (3′iALB) on insulin expression. In comparison with the original promoter, glucose responsiveness was only observed in the modified promoters in vitro with a 36 h lag time before the peak expression. A 50% decrease in the number of viral particles at 5 × 109 vector genome (vg)/mouse was required by AAV8-R3GHLP-hINSco to reduce the blood sugar level to near normoglycemia when compared to the original AAV8-HLP-hINSco that needed 1 × 1010 vg/mouse. The further inclusion of an 860 base-pairs 3′iALB enhancer component in the 3′ untranslated region increased the in vitro gene expression significantly but this increase was not observed when the packaged virus was systemically injected in vivo. The addition of R3G to the HLP promoter in the AAV8-human insulin vector increased the insulin expression and secretion, thereby lowering the required dosage for basal insulin treatment. This in turn reduces the risk of liver toxicity and cost of vector production.

Hepatic expression of the human insulin gene reduces glucose levels in vivo in diabetic mice model

2003 ◽  
Vol 29 (4) ◽  
pp. 424-429 ◽  
Author(s):  
F Ajamian ◽  
TG Titok ◽  
EM Suhorada ◽  
TA Ruban ◽  
M Reeben
Keyword(s):  
Human Insulin ◽  
Insulin Gene ◽  
Diabetic Mice ◽  
Mice Model ◽  
Hepatic Expression ◽  
Glucose Levels ◽  
Human Insulin Gene

Analysis of DNA structure in the human insulin gene-linked polymorphic region in vivo

1992 ◽  
Vol 9 (3) ◽  
pp. 221-225 ◽  
Author(s):  
M. C. U. Hammond-Kosack ◽  
M. W. Kilpatrick ◽  
K. Docherty
Keyword(s):  
Cell Line ◽  
Human Insulin ◽  
Hypervariable Region ◽  
Dna Structure ◽  
Insulin Gene ◽  
Chromosomal Dna ◽  
Β Cell ◽  
Polymorphic Region ◽  
Human Insulin Gene

ABSTRACT An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human β-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived β-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehydereactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat β-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.

The human insulin gene-linked polymorphic region adopts a G-quartet structure in chromatin assembled in vitro

1993 ◽  
Vol 10 (2) ◽  
pp. 121-126 ◽  
Author(s):  
M C U Hammond-Kosack ◽  
M W Kilpatrick ◽  
K Docherty
Keyword(s):  
Human Insulin ◽  
Tandem Repeats ◽  
Consensus Sequence ◽  
Repeat Sequence ◽  
Insulin Gene ◽  
Polymorphic Region ◽  
Dna Structures ◽  
Nuclease P1 ◽  
Human Insulin Gene

ABSTRACT The insulin gene-linked polymorphic region (ILPR), located 363 bp upstream of the human insulin gene, is composed of tandem repeats of the consensus sequence ACAGGGGT(G/C)(T/C)GGGG. It has previously been shown that an insulin gene fragment containing the ILPR adopts an altered DNA structure in vitro. Furthermore, oligonucleotides containing the consensus repeat sequence exhibit multiple quadriplex DNA structures. The present study was undertaken to determine whether such altered DNA structures existed within the ILPR when the insulin gene was assembled into chromatin in vitro. Chromatin assembly was achieved using histones and an extract from unfertilized eggs from Xenopus laevis. The presence of altered DNA conformations within the 5′ region of the human insulin gene was investigated using the structural probe nuclease P1. Nuclease P1 recognized multiple distinct sites in the 5′ flanking region of the human insulin gene in naked DNA. Most of these sites disappeared when the recombinant plasmid DNA was treated with histones and unfertilized egg extract. In the assembled DNA, the ILPR appeared as the major site of nuclease P1 hypersensitivity. Fine-mapping of the multiple reactive sites within the ILPR showed a pattern characteristic of G-quartet foldback structures similar to those that have been observed for telomeric DNA.

In Vitro Amplification of the Human Insulin Gene

1990 ◽  
pp. 119-123
Author(s):  
Ryszard Slomski ◽  
Malgorzata Jungerman ◽  
Adam Kraszweski
Keyword(s):  
Human Insulin ◽  
Insulin Gene ◽  
Human Insulin Gene

Lack of association of the polymorphic locus in the 5'-flanking region of the human insulin gene and diabetes in American blacks

Diabetes ◽  
1985 ◽  
Vol 34 (5) ◽  
pp. 433-439 ◽  
Author(s):  
S. Elbein ◽  
P. Rotwein ◽  
M. A. Permutt ◽  
G. I. Bell ◽  
N. Sanz ◽  
...  
Keyword(s):  
Human Insulin ◽  
Polymorphic Locus ◽  
Insulin Gene ◽  
Human Insulin Gene ◽  
Flanking Region ◽  
American Blacks

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

scholarly journals Variation in the sequence and modification state of the human insulin gene flanking regions

1982 ◽  
Vol 10 (7) ◽  
pp. 2225-2240 ◽  
Author(s):  
Axel Ullrich ◽  
Thomas J. Dull ◽  
Alane Gray ◽  
John A. Philips ◽  
Stephan Peter
Keyword(s):  
Human Insulin ◽  
Insulin Gene ◽  
Human Insulin Gene ◽  
Flanking Regions
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