Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models

Abstract

Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry. Transgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with NM treatment, yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.

Keywords: AAV; LamR; cardiac optogenetics; channelrhodopsin-2; gene therapy; iPS-CM; rat heart; sialic acid.

FIGURE 1

In vitro AAV6-mediated transgene expression is superior to the use of AAV1 and AAV9 in rat and human cardiomyocytes. (A) Cardiomyocyte-specific eGFP expression in NRVMs and hiPSC-CMs using AAV1, 6, and 9. AAV9-mediated expression did not exhibit levels of fluorescence above that of autofluorescence in non-infected control cells. Cell nuclei were labeled with DAPI (blue, NRVMS only), AAV-infected cells expressed eGFP (green), and cardiomyocytes were labeled with α-actinin (red); MOI 1000. (B,D) AAV1-mediated and (C,E) AAV6-mediated eGFP expression at four viral doses 5 days post-infection. hiPSC-CMs required viral doses two orders of magnitude greater than NRVMs (MOI 10,000 versus MOI 100) to show threshold eGFP expression. All scale bars are 50 μm and color-enhanced images are shown. (F) Quantification of the dose-dependent increase in eGFP expression in NRVMs and hiPSC-CMs. AAV6-mediated eGFP expression was significantly higher than AAV1-mediated expression at all viral doses. Data are presented as mean ± SEM (n = 3–7 independent samples per group). ^∗Significance level at p < 0.05.

FIGURE 2

In vivo AAV9-mediated mCherry expression in the adult rat heart provides robust, predominantly cardiomyocyte-specific transgene delivery. (A) Systemic delivery of 0.5 × 10¹² viral particles resulted in robust cardiac-specific expression of mCherry in 4 weeks using AAV9, but not AAV1 as measured using radiant efficiency. Other major organs (including brain, liver, and kidney) showed little to no signs of AAV-mediated infection. Scale bars are 500 μm. (B) AAV9-mediated transgene delivery resulted in transmural ChR2-mCherry expression in both the LV and RV free walls. Scale bars are 250 μm. (C) High-resolution images (brightfield and fluorescence) show cardiomyocyte-specific ChR2-mCherry expression using AAV9-mediated delivery. Scale bars are 50 μm.

FIGURE 3

Proposed mechanisms of infectivity for AAV1, 6, and 9. (A) Cell surface N-linked sialic acid has been proposed as the primary receptor for AAV1 and 6 to infect and transduce cells. The removal of sialic acid by neuraminidase (targeting the portion of the glycoprotein indicated by the arrow) is expected to block the AAV1,6-mediated transduction of cells. (B) AAV9-mediated cell infection/transduction has been attributed to two receptors: terminal galactose on cell surface glycoproteins (left panel) and the 37/67 kDa laminin receptor (LamR) (right panel).

FIGURE 4

In vitro desialylation modulates AAV-mediated eGFP expression in NRVMs and hiPSC-CMs. Cardiomyocyte-specific eGFP expression with (+NM, 25 mU/mL) and without (−NM) neuraminidase treatment prior to viral infection in (A) NRVMs at MOI 2000 and (C) hiPSC-CMs at MOI 30,000. Cell nuclei were labeled with DAPI (blue, NRVMs only) and AAV-infected cells expressed eGFP (green). All scale bars are 50 μm and color-enhanced images are shown. Quantification of eGFP expression with and without desialylation in all three serotypes in (B) NRVMs and (D) hiPSC-CMs. eGFP expression mediated by AAV1 and 6 significantly decreased in both cell types, whereas transgene expression mediated by AAV9 significantly increased in hiPS-CMs only. Application of a higher dose of NM (500 mU/mL) in hiPSC-CMs infected with AAV9 resulted in even greater eGFP expression. Data are presented as mean ± SEM (n = 3–7 independent samples per group). ^∗Significance level at p < 0.05.

FIGURE 5

The 37/67 kDa LamR is expressed in the rat heart, but not in cultured NRVMs and hiPSC-CMs. (A) Negative in vitro immunostains of NRVMs and hiPSC-CMs for LamR. Concurrent immunostaining of HeLa cells, serving as a positive in vitro control for LamR. Scale bars are 50 μm. (B) Positive immunostains of adult and neonatal rat hearts. Concurrent immunostaining of breast carcinoma tissue, serving as a positive in vivo control for LamR. (C) Negative controls of tissue (stained with no primary LamR antibody) showed no contribution to the positive stain by non-specific secondary antibody staining. Scale bars in B,C are 3 mm.

FIGURE 6

The 37/67 kDa LamR is expressed in the intact human heart. Western blots of LamR protein in fresh samples from male and female human hearts and from cultured hiPS-CMs corroborate the difference in expression (lack of LamR in the cultured myocytes and abundance in the intact human heart). GAPDH was used as a loading control and for normalization purposes.

FIGURE 7

Robust in vitro and in vivo optogenetic control of the heart. (A) Adenoviral (AdV)-mediated ChR2-eYFP expression and functional measurements in NRVMs (MOI 25) and hiPSC-CMs (MOI 250) 2 days post-infection. Functional measurements were acquired using voltage- (di-4-ANBDQBS) and calcium- (Rhod4) sensitive dyes and example traces with optical pacing are shown. Cell nuclei were labeled with DAPI (blue) and AdV-infected cells expressed eYFP (green). Alpha-actinin staining (red) showed the cardiospecificity of the ChR2-eYFP infection. (B) Strength-duration curves for AAV9-mediated ChR2 expression in hiPSC-CMs. Conditions for infection included MOIs of 50,000–100,000 and NM applications of 500 mU/mL. Black arrows show the effect of NM treatment on lowering irradiance (mW/mm²) requirements; shown are voltage and calcium traces for the case of using MOI 50,000 without and with NM treatment. Data are presented as mean ± SEM (n = 3 per group). (C) AAV9-mediated ChR2-mCherry expression in the intact adult rat heart after 4 weeks results in optically sensitive myocardium in situ (left panel). A 0.8 mm diameter optical fiber was used to optically control electrical activity from the LV free wall as recorded using ECG (middle panel). Optical pacing resulted in an increased heart rate, as well as significant morphological changes in the QRS complex (right panel); the irradiance needed for this point stimulation was substantially higher than in vitro. Spatial scale bars are 50 μm in B,C. Temporal scale bars are as indicated.