Molecular Redox Mechanisms and Gene Therapy of Non-alcoholic Fatty Liver Disease

Molecular redox mechanisms and gene therapy of nonalcoholic fatty liver disease (NAFLD) Introduction • • • Figure 1: Chromatogram that shows the base pair difference in the WT and mutant mGlrx where Cysteine was changed to Serine Figure 5: Gel Electrophoresis displaying the results of the enzymatic digestion of the vector purified WT C23S/C26S Figure 6: Gel Electrophoresis displaying the results of the PCR on the insert and the purification of both the WT and the mutant Figure 7: Sequencing results from single Gibson Assembly clones for both the WT and C23S/C26S mutant kDa Methods Iodixanol 250 150 100 75 Figure 2: Generating the Vector B • unpurified WT C23S/C26S 1kb ladder (1 SA 50 ng ) • Uncut Cut plasmid plasmid PE G Ch lo ro 2- form Ph as e • 1 kb ladder W • Non-alcoholic fatty liver disease (NAFLD) is a very common liver disease that affects millions and can potentially advance to the more dangerous non-alcoholic steatohepatitis (NASH)1. NASH is likely to progress to cirrhosis and liver failure, and is now the second most common reason for liver transplantation in the US, after chronic viral hepatitis1. The precise cellular and molecular mechanisms underlying the development and progression of NASH remain unknown and there are no effective FDA-approved therapies for NASH. Thus, there is an urgent need for innovative strategies, propelled by promising findings in mouse models, to stop or even reverse NASH. Glutaredoxin-1 (Glrx) is a small redox enzyme which is abundant in the liver, and data of the Bachschmid laboratory shows that a Glrx deficiency causes fatty liver in mice, similar to patients with NAFLD where hepatic Glrx expression is decreased with increased protein glutathionylation2. We hypothesized that an overexpression of Glrx caused by an injection of an engineered adeno associated virus (AAV) would stop and reverse the effects of fibrosis. AAVs are a viral gene therapy delivery method that has been shown to work quickly, have high gene transfer efficiency, and have stable long term expression. AAVs are also safer for the patient than regular adeno virus. We used the DJ serotype due to its suitability for cell culture and in vivo applications. As we are testing the effectiveness of wild type mouse Glutaredoxin (mGlrx), we compared it to the a mutant mGlrx, C23S/C26S. This mutant’s amino acid sequence differs from that of the WT Glrx because it has two cysteines in the sequence that have been changed to serines, an amino acid change that basically deactivates the mGlrx entirely. This served as a control for the experiment. Our virus was tested on male C57BL/6J mice which have been fed a high fat, high fructose, high cholesterol diet which has been shown to induce liver fibrosis faster than any previous diet, as well as on a control group which was fed standard chow. M • Results Viral bands 50 37 25 1 2 3 4 5 Fraction Grow bacteria with plasmid Miniprep an overnight pAAV.TBG.PI.eGFP.WPRE.bGH culture of a single colony to extract DNA Run enzymatic digestion with NotI and HindIII to remove EGFP and then heat inactivate the enzymes Figure 8: Plasmid map of the WT construct created in the Gibson Assembly Purify cut DNA product Figure 9: The collected viral samples loaded onto a protein gel to determine purity compared to BSA standards. Figure 3: Gibson Assembly Cloning Discussion/ Conclusion Digested vector after enzyme heat inactivation PCR amplified WT or mutant mouse glutaredoxin (mGlrx) insert that has been purified • Vector, insert, primers, and Gibson Assembly Master Mix are added together Cloning process occurs over an hour long incubation period at 50℃ Result is shocked into competent NTI stable cells, plated, grown, and maxi prepped to extract recombinant DNA • • Figure 4: Cell Culture and Virus Extraction • Culture HEK293T cells Transfect cells with growth plasmid (pHelper), capsid plasmid (pRepCap), and the recombinant plasmid that was created from Gibson Assembly Collect virus from cell medium through lysing, titration, and purification Inject animals • Figure 5 shows that the digested plasmid has two bands, one at around 700 base pairs and one of around 4900 base pairs. This proves that the EGFP which was in the original plasmid had been cut out as it is 729 base pairs. The other uncut plasmid serves as the control and we are able to confirm that the digestion actually occurred as opposed to a random folding of the DNA that mimics the expected result Both types of mGlrx inserts, WT and C23S/C26S, were amplified with PCR and purified through silica columns to remove any background. Figure 6 shows that after purification both types of insert were more concentrated and brighter on the gel as well as their being less background. The high concentration of the insert was important because for the Gibson Assembly, the insert and the plasmid were added in a 4:1 ratio to increase the chances of the assembly succeeding After the Gibson assembly was performed, and its results were confirmed by sequencing. Figure 7 shows the sequencing results of a maxiprep done on an overnight culture of a single clone colony. The results show that in both the WT and the mutant cases, the insert was successfully added into the vector. Figure 8 is a visual representation of what the complete vector looks like, including all of it’s tags, promoters, and important restriction sites. Figure 9 shows the titration gradient of the virus after it was collected rom the transfected cell media after 3 days. As can be seen, fractions 2, 3, and 4 are relatively pure but fraction 5 has a low protein band which could cause an inflammatory response when injected into the animals. The actual effectivity of the virus will be unknown until expression can be detected in the mice which will take time after the animals are injected