Jacobs, F, Snoeys, J, Feng, Y, Van Craeyveld, E, Lievens, J, Armentano, D et al.. Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer. Gene Ther 15: 594-603
Hepatocytes are a key target for treatment of inborn errors of metabolism, dyslipidemia and coagulation disorders. The development of potent expression cassettes is a critical target to improve the therapeutic index of gene transfer vectors. Here we evaluated 22 hepatocyte-specific expression cassettes containing a human apo A-I transgene following hydrodynamic transfer of plasmids or adenoviral transfer with E1E3E4-deleted vectors in C57BL/6 mice. The DC172 promoter consisting of a 890 bp human alpha(1)-antitrypsin promoter and two copies of the 160 bp alpha(1)-microglobulin enhancer results in superior expression levels compared to constructs containing the 1.5 kb human alpha(1)-antitrypsin promoter, the 790 bp synthetic liver-specific promoter or the DC190 promoter containing a 520 bp human albumin promoter and two copies of the 99 bp prothrombin enhancer. The most potent expression cassette consists of the DC172 promoter upstream of the transgene and two copies of the hepatic control region-1. Minicircles containing this expression cassette induce persistent physiological human apo A-I or human factor IX levels after hydrodynamic transfer. In conclusion, in this comparative study of 22 hepatocyte-specific expression cassettes, the DC172 promoter in combination with two copies of the hepatic control region-1 induces the highest expression levels following hydrodynamic and adenoviral transfer.
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"The efficiency of Gene therapy depends on successful delivery of candidate gene to specific and correct locus in the genome. While carrying out gene therapy at the clinical level, it becomes more important to regulate the transformation of fresh copy in a required copy number without altering the rest of the genome (Jacobs et al., 2008). Gene transformation efficiency and its expression are other parameters which define the efficiency of gene therapy. "
[Show abstract][Hide abstract] ABSTRACT: The twentieth century is considered for the technological revolution in different fields such as Industry, Research and Medicine. Scientific inventions have improved Research and Industrial output while medicine one step ahead. Numerous inventions have made a revolution in the management of life threatening diseases which were incurable decades ago. Due to these revelations, health care system is growing exponentially and especially, Gene Therapy is known as one of the most advanced approaches for the treatment of diseases associated with abnormal functioning of the genome. Gene therapy offers management of diseases/disorders through manipulation at genetic level either by replacement of abnormal gene(s) and /or repairs. Gene therapy essentially requires targeted and efficient gene delivery to tissue or cell. There are numerous methods available to carry out gene delivery either In-Vivo or Ex-Vivo for particular diseases. Both viral and non-viral vehicles used for delivery of exogenous genes have shown tremendous benefits in numerous clinical trials carried out over last few decades. Both the options, viral and non-viral tools, for gene delivery with remarkable significance are often linked with numerous complications. To surpass these complications, novel tools like Nano-constructs designed for nano-materials are in practice and have shown promising results in delivering candidate drugs and biomolecules. Here in this review, we have summarized the potential of new generation delivery vehicles and their advantages over viral and other available non-viral vehicles. Also, current information with respect to the design and functioning of Nano-constructs implemented in clinical study for management of many diseases is provided.
"Generation of recombinant adenoviral vectors The E1E3E4-deleted adenoviral vector AdLDLr contains the 1.2 kb DC172 promoter , consisting of an 890 bp human α1-antitrypsin promoter and two copies of the 160 bp α1-microglobulin enhancer, upstream of the 5′ untranslated region (UTR) of the human apo A-I gene that contains the first intron, and upstream of the 2.6 kb LDLr sequence  and two copies of the 774 bp hepatic control region-1. Following an EcoRI restriction, the 5′ UTR of the human apo A-I gene together with the 2.6 kb LDLr cDNA sequence was isolated from pGEM-A-Iintron-LDLr  and subsequently ligated into the EcoRI site of pTG15203-DC172.HCR2 . Construction of the E1E3E4-deleted adenoviral vector AdA-I containing the DC172 promoter upstream of the genomic human apo A-I sequence and two copies of HCR-1 has been described before . "
[Show abstract][Hide abstract] ABSTRACT: Both reductions in atherogenic lipoproteins and increases in high-density lipoprotein (HDL) levels may affect atherosclerosis regression. Here, the relative potential of low-density lipoprotein (LDL) lowering and HDL raising gene transfer strategies to induce regression of complex murine atherosclerotic lesions was directly compared. Male C57BL/6 LDL receptor (LDLr)−/− mice were fed an atherogenic diet (1.25% cholesterol and 10% coconut oil) to induce advanced atherosclerotic lesions. A baseline group was killed after 6 months and remaining mice were randomized into a control progression (Adnull or saline), an apolipoprotein (apo) A-I (AdA-I), an LDLr (AdLDLr), or a combined apo A-I/LDLr (AdA-I/AdLDLr) adenoviral gene transfer group and followed-up for another 12 weeks with continuation of the atherogenic diet. Gene transfer with AdLDLr decreased non-HDL cholesterol levels persistently by 95% (p < 0.001) compared with baseline. This drastic reduction of non-HDL cholesterol levels induced lesion regression by 28% (p < 0.001) in the aortic root and by 25% (p < 0.05) in the brachiocephalic artery at 12 weeks after transfer. Change in lesion size was accompanied by enhanced plaque stability, as evidenced by increased collagen content, reduced lesional macrophage content, a drastic reduction of necrotic core area, and decreased expression of inflammatory genes. Elevated HDL cholesterol following AdA-I transfer increased collagen content in lesions, but did not induce regression. Apo A-I gene transfer on top of AdLDLr transfer resulted in additive effects, particularly on inflammatory gene expression. In conclusion, drastic lipid lowering induced by a powerful gene transfer strategy leads to pronounced regression and stabilization of advanced murine atherosclerosis.
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Preview · Article · Jun 2011 · Journal of Molecular Medicine
"Importantly, the greater efficacy of such double-strand vectors compared to ssAAV will enable lower doses to be used with concomitant benefits in limiting hepatotoxicity, immunogenicity and non-hepatic expression. These are significant considerations and give impetus for introducing further vector refinement, including apoE3 codon optimization   or improved transcriptional regulatory elements  . "
[Show abstract][Hide abstract] ABSTRACT: Hepatic gene transfer of atheroprotective human apoE by recombinant viral vectors can reverse hypercholesterolaemia and inhibit atherogenesis in apoE-deficient (apoE(-/-)) mice. Here, in preliminary studies we assess the effectiveness of a recently developed self-complementary adeno-associated virus (scAAV) serotype 8 vector, driven by a hepatocyte-specific promoter (LP1), for liver-directed gene delivery of human apoE3. Vector viability was validated by transducing cultured HepG2 cells and measuring secretion of apoE3 protein. Male and female apoE(-/-) mice, 6-month old and fed on normal chow, were intravenously injected with 1x10(11) vg (vector genomes) of scAAV2/8.LP1.apoE3; age-matched untreated mice served as controls. In male mice, plasma apoE3 levels were sufficiently high (up to 17 microg/ml) to normalize plasma total cholesterol and ameliorate their proatherogenic lipoprotein profile, by reducing VLDL/LDL and increasing HDL 5-fold. At termination (12 weeks) development of aortic atherosclerosis was significantly retarded by 58% (aortic lesion area 8.2+/-1.4% vs. 19.3+/-2.4% in control males; P<0.001). Qualitatively similar anti-atherogenic effects were noted when female mice were treated, but the benefits were less marked and aortic lesions, for example, were reduced by only 33% (15.7+/-3.7% vs. 23.6+/-6.9%). Although group numbers were small (n=4/5), this gender-specific difference reflected two to three times less apoE3 in plasma of female mice at weeks 3 and 6, implying that gene transfer to female liver using scAAV vectors may require additional optimization, despite their established superior potency to conventional single-stranded (ssAAV) vectors.