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Introduction: A high transduction efficacy and potency of AAV-based cardiac gene therapies is key for the clinical translation in which hereditary and acquired cardiac disorders will be targeted. As such, ultrapure vectors with superior biological and therapeutic capabilities are a must for these therapies. Hypothesis: We hypothesized that our developed affinity chromatography (AC) based purification system will increase AAV recovery and - potency. Methods: AAV vector production, AC based - or iodixanol density gradient (DG) based purification, Q-PCR, WB, EM, LC-MS/MS, P-loop Results: Using the same vector input quantity, the AC-based purification enabled an approximately 13-fold greater vector genome copy (vgc) recovery than the DG-based purification. Mass spectrometry analysis demonstrated ultrapure AAV9 vectors as a result of an AC purification (AAV9 93.24% vs. 6,76% contaminants) whereas corresponding DG preparations resulted in highly contaminated vectors (AAV9 5.49% vs. 94,51% contaminants). Biological potency of AC- and DG-purified AAV9 vectors towards cardiac transduction were determined by systemic injections of 1·10 10 , 1·10 11 or 1·10 12 vgc of AAV9-EGFP in C57BL/6 mice (n=8 each group). AC-purified AAVs achieved a significantly higher cardiac transduction efficacy for every dosage assessed by comparative bulk myocardial DNA, RNA and protein level analysis after 2 weeks. Therapeutic potency was examined for a recently published novel target for chronic heart failure namely the RFXP1-RLN system. To this end, a dosage of 5·10 11 vgcs of either AC- or DG purified AAV9-RXFP1 vectors were systemically injected and the cardiac contractile performance increase was captured after 2 weeks in mice of both groups. Of note, 10 minutes after RLN administration, the rise in LV +dp/dt max. was already significantly greater in the AC- than the DG-vector treated group (AC: 13594+/-1972 vs. DC: 9822+/-801 mmHg/s; n=8 per group, p Conclusion: The data clearly promote AC-based AAV purification as a novel standard for cardiovascular basic and translational research. Higher consistency in results, higher therapeutic effects and superior biological potency can be expected from higher production yields of ultrapure AAVs even at lower vector dosages.

Simple Summary Hepatocellular carcinoma (HCC) represents the most common type of liver cancer and has a poor prognosis. Therefore, there is an urgent need for the identification of new therapeutic options. The mouse double minute homolog 4 (MDM4) gene, a known p53 inhibitor, is upregulated in most HCCs. Here, we aimed to investigate the mechanisms leading to MDM4 transcriptional upregulation and to evaluate whether therapeutic targeting of these mechanisms might represent a suitable approach for future therapy. Using human HCC cell lines, a mouse model, and human HCC cohorts, we have identified serum response factor (SRF), ETS transcription factors ELK1 and ELK4 as transcription factors (TFs) driving MDM4 expression. Treatment of HCC cell lines with XI-011, a pharmaceutical inhibitor of MDM4 transcription, reduced the expression of both the TFs and MDM4 and impaired tumor growth, suggesting that targeting the MDM4 transcription may provide a rationale for future targeted therapy of HCC. Abstract Different molecular mechanisms support the overexpression of the mouse double minute homolog 4 (MDM4), a functional p53 inhibitor, in human hepatocellular carcinoma (HCC). However, the transcription factors (TFs) leading to its transcriptional upregulation remain unknown. Following promoter and gene expression analyses, putative TFs were investigated using gene-specific siRNAs, cDNAs, luciferase reporter assays, chromatin immunoprecipitation, and XI-011 drug treatment in vitro. Additionally, MDM4 expression was investigated in SRF-VP16iHep transgenic mice. We observed a copy-number-independent upregulation of MDM4 in human HCCs. Serum response factor (SRF), ELK1 and ELK4 were identified as TFs activating MDM4 transcription. While SRF was constitutively detected in TF complexes at the MDM4 promoter, presence of ELK1 and ELK4 was cell-type dependent. Furthermore, MDM4 was upregulated in SRF-VP16-driven murine liver tumors. The pharmacological inhibitor XI-011 exhibited anti-MDM4 activity by downregulating the TFs driving MDM4 transcription, which decreased HCC cell viability and increased apoptosis. In conclusion, SRF drives transcriptional MDM4 upregulation in HCC, acting in concert with either ELK1 or ELK4. The transcriptional regulation of MDM4 may be a promising target for precision oncology of human HCC, as XI-011 treatment exerts anti-MDM4 activity independent from the MDM4 copy number and the p53 status.