AAV Vector Toolkit for the Delivery and Expression of the Artificial microRNA in the Murine Heart

Figure 1. AAV-mediated transgene expression in the murine heart and liver under different administration conditions. The adult wild-type mice were treated with the AAV9-CMV-eGFP vector by indirect IC (5 × 1011 GC/animal), IM (5 × 1011 GC/animal), and IV (4 × 1012 GC/animal) injections (n = 5 animals per treatment). Four weeks post-injection, eGFP expression in the heart and liver sections was analyzed by fluorescent microscopy. (a) Representative images of heart sections following each injection condition are shown. Scale bar: 1 mm. (b) Semi-quantitative evaluation of relative eGFP signal intensity of the heart sections after IC (n = 1 sample), IM (n = 2), and IV (n = 2) injections. (c) Representative images of liver sections are shown. Scale bar: 1 mm.

Figure 2. Direct comparison of MyoAAV 2A and 4E serotypes with parental AAV9 for heart-liver transduction properties. The 8-week-old mdx mice were systemically injected with AAV9-CMV-eGFP, MyoAAV 2A-CMV-eGFP, and MyoAAV 4E-CMV-eGFP vectors at 8 × 1013 GC/kg (n = 3 animals per treatment). Four weeks post-injection, eGFP fluorescence (n = 1) and mRNA levels (n = 2) were evaluated. Cryosections of mouse hearts were analyzed by fluorescent microscopy. (a) Representative images show transduction of murine myocardium by serotypes AAV9, MyoAAV 2A, and MyoAAV 4E. Scale bar: 30 μm. (b) The expression of eGFP mRNA in muscles and liver was quantified by RT-qPCR and presented as a muscle-to-liver ratio for each animal. Each data point represents one sample.

Figure 3. In vitro evaluation of commonly used cardiac and pan-muscular promoters to drive efficient and selective expression in cardiomyocytes. (a) Schematically shown is the AAV expression vector with the reporter eGFP under the control of one of the selected promoters. CMV, human cytomegalovirus immediate-early enhancer and promoter; αMHC, promoter of α-myosin heavy chain; cTnT, promoter of chicken cardiac troponin T gene; NCX1, promoter of feline cardiac Na+/Ca2+ transporter; MHCK7, hybrid promoter, conjugate of creatinine kinase regulatory region and α-myosin heavy chain enhancer; SPc5-12, synthetic promoter, combination of SRE, TEF-1, MEF-1, and MEF-2 regulatory sites; CK8e, hybrid promoter based on muscle creatinine kinase gene; mDes, murine desmin promoter; hDes, human desmin promoter; CRM4-mDes, murine desmin promoter with CRM4 enhancer. The size in base pairs is indicated for each promoter. (b) Patient-specific hiPSC-CMs (line IPSFIL14S) served as an in vitro model for screening promoter activity. hiPSC-CMs were co-immunostained for the cardiac marker cTnT and cytoskeleton protein F-actin; nuclei were counterstained with DAPI (blue). Images demonstrate colocalization of cTnT and…

Figure 4. The activity of cardiac and muscular promoters in the heart and skeletal muscles following systemic MyoAAV 2A-mediated delivery. Adult mice were intravenously injected with 4 × 1013 GC/kg of MyoAAV 2A vectors carrying eGFP reporters under cTnT, CK8e, or CRM4-mDes promoters and analyzed four weeks after injection. (a) MyoAAV 2A vector biodistribution in murine tissues. Data presented as mean ± SD, n = 5 animals per group. For all vectors, the difference in vector distribution between similar tissues was statistically insignificant, as established by the Mann–Whitney test. (b) eGFP fluorescence in different tissues of AAV-treated mice illuminated by blue light. (c) The relative promoter activity calculated as eGFP mRNA expression quantified by RT-qPCR (see Materials and Methods and Figure S8a), normalized to the AAV vector DNA; the promoter activity in the liver was set as 1. (d) The relative promoter activity calculated as eGFP fluorescence measured in the tissue lysates by the microplate assay, normalized to AAV vector DNA. Data presented as organ-to-liver ratio. * p < 0.05 (Mann–Whitney test).

Figure 5. The artificial miRNA is expressed and processed in murine hearts following systemic injection of the MyoAAV 2A-cTNT-eGFP-amiR-A12.1 vector. (a) Schematically shown is the MyoAAV 2A vector carrying the expression cassette cTnT-eGFP-amiR-A12.1, which enables cTnT-driven synthesis of the protein-coding transgene (eGFP) and miR-30-based artificial miRNA (amiR-A12.1). The MyoAAV 2A-cTNT-eGFP-amiR-A12.1 vector was IV injected into adult, wild-type mice at 2 × 1014 GC/kg; n = 4 for the vector or vehicle (DPBS) group. (b) MyoAAV 2A vector biodistribution in murine tissues one month post-injection. (c) Expression of the 22-mer mature guide strand of amiR-A12.1 quantified by stem-loop RT-qPCR in the heart and liver. (d) Expression of eGFP mRNA quantified by RT-qPCR. Data were presented as individual data points with mean ± SD. Statistical significance was determined using the Mann–Whitney test.