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Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.

BackgroundGenomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the site which is disrupted upon integration of Adeno Associated Virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in Progressive Familial Intra Hepatic Cholestasis Type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient.MethodsWe used CRISPR Cas9 a genome editing tool to insert ABCB11 gene at AAVS1 site in human cell-lines.ResultsWe found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli and the removal of the same was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that ABCB11 protein has similarity with E. coli Endotoxin (Lipid A) Transporter MsbA.ConclusionWe inserted ABCB11 at AAVS1 site using CRISPR-Cas9, however, the frequency of homologous recombination was very low for this approach to be successful in-vivo (Figure: pictorial abstract).Pictorial AbstractABCB11 gene (which codes the transporter of human bile salts) is targeted to AAVS1 site using a construct which has 5’ and 3’ overhangs which are homologous to the AAVS1 site. A Pribnow box was detected inside ABCB11 gene which allowed the gene to transcribe in E. Coli causing bacterial lysis probably through competitive replacement of a homologous transporter protein in E. Coli (E. coli Endotoxin (Lipid A) Transporter) MsbA, resulting in Lipid A (L) accumulation inside the bacteria.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2/2019-nCoV) infection is an emerging pandemic. The virus binds to angiotensin converting enzyme 2 (ACE2) and TMPRSS2 which are abundantly expressed on various human cells including lung epithelial cells and intestinal cells and the virus can infect these cells. Currently no specific treatments or vaccines are available for this disease. A per oral live attenuated vaccine can be beneficial in SARS-Cov-2 infection because the attenuated virus initially infects the gut, stimulates the mucosa associated immune system sparing the respiratory system during the initial immune response. The live virus can also spread in the community boosting herd immunity.

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.

ObjectiveTo study the role of miRNA-181a and augmenter of liver regeneration in TGF-β-induced fibrosis in hepatic stellate cells.MethodsLX2 cells were treated with 20 ng/ml TGF-β for 24 h. miRNA-181a, ALR plasmid and empty vectors were transfected using siPORT NeoFx reagent. Cells were harvested after 48 h or 72 h of transfection for protein or RNA analysis. Western blotting was performed for ALR, TGF-β receptor II (TGFβ-RII), collagen 1A1 (COLL1A1), alpha-smooth muscle cell actin (α-SMA), rac1, E-cadherin and β-actin. Quantitative RT-PCR was performed for ALR, GAPDH, miRNA-181a or 5S rRNA.ResultsTGF-β induced the expression of miRNA-181a, which in turn down-regulated ALR thereby induced the fibrosis markers, such as COLL1A1, α-SMA and rac1 in hepatic stellate cells. Over-expression of miRNA-181a down-regulated expression of ALR and up-regulated expression of fibrosis markers. On the other hand, ALR over-expression resulted in a decrease in miRNA-181a expression and fibrosis markers. Over-expression of ALR also inhibited the expression of TGFβ-RII and increased expression E-cadherin.ConclusionTGF-β induced miRNA-181a, which in turn induced fibrosis, at least in part, by inhibiting ALR. ALR inhibited TGF-β action by decreasing the expression of TGFβ-RII, thereby inhibiting miRNA-181a expression and fibrosis markers. ALR could serve as a potential molecule to inhibit liver fibrosis.

Potential of nonantibiotic therapies for treatment of Helicobacter pylori-related acid peptic disease remains underexplored. Several clinical studies have shown that higher prevalence of H. pylori infection is associated with low Vitamin C (Vit C) level in serum and gastric juice. However, there is no consensus regarding the usefulness of Vit C supplementation in the management of H. pylori infection. Surveying the existing literature we conclude that high concentration of Vit C in gastric juice might inactivate H. pylori urease, the key enzyme for the pathogen's survival and colonization into acidic stomach. Once infection established, urease is not very important for its survival. The role of Vit-C as anti-H. pylori agent in peptic ulcer diseases appears to be preventive rather than curative. Rather than supplementing high dose of Vit C along with conventional triple therapy, it is preferable to complete the conventional therapy and thereafter start Vit C supplementation for extended period which would prevent reinfection in susceptible individuals, provided the patients are not achlorhydric. Further studies are required to prove the role of Vit C in susceptible population.

Nonalcoholic fatty liver disease (NAFLD) is a group of diseases with excess fat in liver in the absence of a poorly defined limit of alcohol consumption. Most common variety, a universal public health problem, is associated with insulin resistance caused by a host of genetic and epigenetic defects modulated by life style and environmental factors. In fact the term NAFLD is loose to incorporate so many etiologies except alcoholism and few other etiologies, presenting as fat in liver. However as a sign fatty liver is very important in predicting the risk of diabetes, cardiovascular disease, stroke, cirrhosis and cancer. Abnormal fat accumulation can result from several defects in nuclear receptors associated with lipid sensing, synthesis and oxidation like LXR, FXR, SREBP, ChREBP and PPAR; defects in the lipid influx-efflux channels, insulin signaling, proteins involved in fatty acid catabolism, defects in adipose tissue development and function, inappropriate nutrition and finally defects in neural regulatory mechanisms. The progress of the disease is determined by the basic defects which results in fat accumulation, an individual’s immunological response to the accumulated fat and its derivatives and the oxidant stress response. Congregation of unrelated genetic defects under same diagnosis ‘NAFLD’ can result in inefficient patient management. Further studies are required to understand the molecular basis of fatty liver to enable a personalized management of diseases presenting as fatty liver in the absence of alcohol abuse.

Strategies to fill the huge gap in supply versus demand of human organs include bioartificial organs, growing humanized organs in animals, cell therapy, and implantable bioengineered constructs. Reproducing the complex relations between different cell types, generation of adequate vasculature, and immunological complications are road blocks in generation of bioengineered organs, while immunological complications limit the use of humanized organs produced in animals. Recent developments in induced pluripotent stem cell (iPSC) biology offer a possibility of generating human, patient-specific organs in non-human primates (NHP) using patient-derived iPSC and NHP-derived iPSC lacking the critical developmental genes for the organ of interest complementing a NHP tetraploid embryo. The organ derived in this way will have the same human leukocyte antigen (HLA) profile as the patient. This approach can be curative in genetic disorders as this offers the possibility of gene manipulation and correction of the patient's genome at the iPSC stage before tetraploid complementation. The process of generation of patient-specific organs such as the liver in this way has the great advantage of making use of the natural signaling cascades in the natural milieu probably resulting in organs of great quality for transplantation. However, the inexorable scientific developments in this direction involve several social issues and hence we need to educate and prepare society in advance to accept the revolutionary consequences, good, bad and ugly.

Fatty liver is present in over ten percentage of the world population and it is a growing public health problem. Nonalcoholic fatty liver disease (NAFLD) is not a single disease, but encompasses a spectrum of diseases of different etiologies. It is difficult to find highly specific and sensitive diagnostic biomarkers when a disease is very complex. Therefore, we should aim to find relevant prognostic markers rather than accurate diagnostic markers which will help to minimize the frequency of liver biopsies to evaluate disease progression. There are several biomarker panels commercially available, however, there is no clear evidence that more sophisticated panels are better compared to simple criteria such as, presence of diabetes over five years, metabolic syndrome, obesity, obstructive sleep apnea, aspartate transaminase/alanine transaminase (ALT) ratio > 0.8 or ferritin levels > 1.5 times normal in patients with over six month history of raised ALT and/or ultrasonological evidence of fat in the liver. Currently the biomarker panels are not a replacement for a liver biopsy. However the need and benefit of liver biopsy in NAFLD is questionable because there is no convincing evidence that biopsy and detailed staging of NAFLD improves the management of NAFLD and benefits the patient. After all there is no evidence based treatment for NAFLD other than management of lifestyle and components of “metabolic syndrome”.