Using human organoids for selecting AAV capsids

This article is based on a poster originally authored by Yingying Fu, Zhen Qi, Zhanguang Zuo, Spencer Chiang, An Ouyang, Glory Gao, Shuge Guan, Jessie Chen, Rosanna Zhang and Cheng Wang from ACROBiosystems.

Organoids represent a promising advance in disease modeling and drug development, helping to reduce clinical drug failure, which is common even after stringent preclinical testing. Current organoid technologies typically lack certain physiological cell types, translatable biological assays and standardized protocols, however.¹

Despite these limitations, organoids are widely recognized as a gold standard preclinical drug development model. Their translatable human genomic background mimics the three-dimensional architecture and function of their respective organs while offering excellent potential for high throughput.²

The use of organoids as preclinical models has the potential to considerably enhance drug development pipeline efficiency, reducing dependence on animal models that are often unable to accurately predict human responses.

ACROBiosystems has developed novel hydrogel-free human iPSC-derived, assay ready, cardiac and cerebral organoid systems. These systems are now commercially available.

Both cerebral and cardiac organoids bear several cell types. These cell types can be verified via marker immunolabeling and RNA-seq, while demonstrating the functional characteristics of their respective organs. These models can also be verified via MEA, patch clamp, and silicon probe recording.

A Parkinson’s disease model was also created via the addition of alpha-synuclein pre-formed fibrils to cerebral organoids. The cardiac organoid was utilized as a model to enable testing of a drug’s cardiac toxicity.

It was noted that both organoids represented promising models for AAV screening in the field of gene therapy development. ACROBiosystems’ advances in this area are anticipated to improve guidance for disease and toxicity modeling, as well as enhancing gene therapy development when progressing into clinical research.

Methods and materials

iPSC and organoid generation

Organoids were created using commercially available human iPSCs (Cat. No.: ACS-1007, ATCC). This was done using either ACROBiosystems’ Cerebral or Cardiac organoid kit (Cat. No. RIPO-BWM001K, RIPO-HWM002K, respectively) in line with the protocol outlined in the relevant data sheet.

Harvested iPSCs were seeded onto a 96-well ultra-low attachment plate. EB formation was monitored, and cells were treated with appropriate media (provided in the kit) in order to trigger organoid formation and subsequent cell differentiation.

The resulting organoids were utilized for assays on the specified timepoints. In order to enable cell marker confirmation, organoids were fixed, permeabilized and immunolabeled with the specified cell markers.

In the case of the Parkinson’s disease model, cerebral organoids were treated on day 92 with 0.1 μM and 1 μM alpha-synuclein pre-formed fibrils (Cat. No. ALN-H5115) for a total of 12 days.

In the case of the cardiac toxicity model, cardiac organoids were treated on day 25 with varying concentrations of a confidential drug referred to as ‘drug A.’

Finally, AAV transduction was performed by transducing cerebral organoids (at 101 days) and cardiac organoids (at 11 days) with 1011 vg of eGFP transgene-bearing AAV5 WT as a negative control or using optimized AAV serotypes containing IVB-1 and IVB-2.

Organoids were incubated at 37 °C 5 % CO₂ in all cases. This was done while shaking the organoids at 100 rpm until these were ready to be analyzed.

Results

Characterization of cerebral organoids: Cell marker verification

Human iPSC-derived cerebral organoids were produced using a Cerebral Organoid Differentiation kit (Figure 1A). These were characterized using immunolabeling. This was performed on various days of culturing (Figures 1B-E):

• On day 36 to investigate neural progenitor and immature neuronal cells
• On day 109 to investigate dopaminergic neurons
• On day 92 to investigate mature neurons
• On day 109 to investigate astrocytes
• On day 119 to investigate oligodendrocytes
• On day 147 to investigate for microglia (day 147)

Figure 1. (A) Cerebral organoid differentiation timeline. Immunofluorescence confirmation of (B) NESTIN and TUJ1, (C) MAP2 and TH, (D) GFAP, (E) OLIG2 and (F) IBA1 positive cells on different stages of organoid maturation. Image Credit: ACROBiosystems

Characterization of cerebral organoids: Transcriptomic and functional characterization

Cerebral organoids exhibit a number of neuronal subtypes and glial cell types. These can be verified via bulk RNA-seq (Figure 2A).

Cerebral organoids demonstrated extracellular electrical activity. This can be verified via MEA analysis (Figure 2B) and silicon probe recording (Figure 2C). Silicon probe recording also highlighted several neuronal groups in early network activity (Figure 2D), while intracellular electrical activity was verified via patch clamp.

Figure 2 . (A) Transcriptome profiling of cerebral organoids. Extracellular electrical activity by (B) MEA and (C) silicon probe recording. (D) Intracellular electrical activity by patch clamp. Image Credit: ACROBiosystems

Parkinson’s disease and AAV capsid screening modeling on cerebral organoids

It was observed that treating healthy 92-day-old cerebral organoids with alpha-synuclein pre-formed fibrils for a total of 12 days (Figure 3A, left) can trigger dopaminergic neuron degeneration and the disruption of other neurons’ dendrites in a concentration-dependent manner (Figure 3A, right).

Cerebral organoids were grown for 101 days before being infected with AAV5-WT, IVB-1, and IVB-2. It was noted that the IVB-2 subtype demonstrated higher infectiousness and transgene delivery towards cerebral organoids.

Figure 3. Cerebral organoids as models for (A) Parkinson’s disease and (B) AAV capsid screening. Image Credit: ACROBiosystems

Characterization of cardiac organoids: Cell marker verification

Human iPSC-derived cardiac organoids were produced using Cardiac Organoid Differentiation kits (Figure 4A). Immunolabeling was used to characterize these organoids for cardiomyocytes on day 12 (Figure 4B) and mechanical beating (Figure 4C, beating not shown).

It was observed that in cardiac organoids, mechanical beating became visible after day 9 of culturing. Calcium sensor imaging confirmed the presence of calcium flux behavior on human heart organoids (Figure 4D).

From a morphological perspective, the cardiac organoids analyzed presented ventricular and atrial-like chambers on different regions. These were also validated using ventricular and atrial cardiomyocyte markers (Figure 4E). The presence of endothelial cells was also confirmed on the cardiac organoids (Figure 4F).

Figure 4. (A) Cardiac organoid differentiation steps. Immunofluorescence confirmation of (B) cTNT and (C) visualization of cardiac organoids on tissue culture plates. (D) Sequential images of calcium sensor imaging of cardiac organoids. (E) Presence of morphological and marker validated atrial (MLC2 A+ cells) and ventricular (MLC2V+ cells) chambers. (F) Vascularization was confirmed by CD31 positive endothelial cell immunolabeling. Image Credit: ACROBiosystems

Characterization of cardiac organoids: Functional characterization

The analyzed heart tissue was determined to be electrically active, with electrical activity probed using MEA recordings. These recordings confirmed that the beating periods and amplitudes of the cardiac organoid model were consistent with three other cardiac models over periods up to 6 hours (Figure 5A).

Silicon probe recordings can also be used to measure extracellular electrical activity. These measurements confirmed physiological wave complex elements for heart activity (Figure 5B). Patch clamp was also used to verify intracellular electrical activity, enabling the identification of a physiological cardiac waveform (Figure 5C).

Figure 5. (A) Electrical activity of the cardiac organoids were confirmed extracellularly by (A) MEA and (B) silicon probe recordings and intracellularly by (C) patch clamp recordings. The resemblance of a physiological waveform of cardiac beating was identified. Image Credit: ACROBiosystems

Cardiac toxicity and AAV capsid screening modeling of cardiac organoids

It was observed that treatment of healthy 25-day-old cardiac organoids with an appropriate concentration of a confidential drug referred to as ‘drug A’ led to degeneration of cardiomyocytes and endothelial cells (Figure 6A).

Eleven-day-old cardiac organoids were infected with AAV5-WT, IVB-1, and IVB-2. It was noted that the IVB-2 subtype demonstrated higher infectiousness and transgene delivery towards cardiac organoids. It was possible to visualize this behavior using fluorescent intensity from GFP transgene expression.

Figure 6. (A) Cardiac toxicity model by Drug A treatment on cardiac organoids. (B) Cardiac organoids as models for AAV capsid screening. Image Credit: ACROBiosystems

Conclusion

Even after rigorous preclinical testing, pharmaceutical drug development experiences high rates of clinical failure due to efficacy issues and side effects. These failures can frequently be attributed to a lack of relevant and translatable cell models.

Organoids are three-dimensional in vitro tissue models able to mimic their respective organs’ architecture and function. Organoids represent a promising advance in drug development and disease modeling, with the potential to considerably reduce clinical drug failure rates.

Current organoid technologies typically lack specific physiological cell types, as well as translatable biological assays. For example, cardiac organoids bear all relevant cell types, show mechanical beating, and are functionally active; while cerebral organoids show functional activity while bearing several neuronal subtypes and all glial cell types.

It was possible to create a Parkinson’s disease model by adding alpha synuclein pre-formed fibrils to cerebral organoids. This recapitulated the dopaminergic neuronal degeneration phenotype.

It was also observed that cardiac organoids degenerated following the addition of a cardiotoxic drug, allowing researchers to observe the cardiomyocyte and endothelial degeneration phenotype.

Both organoids represent promising models for AAV screening as part of gene therapy developments. Cerebral and cardiac organoids were found to be excellent preclinical models for drug toxicity analysis, drug screening, disease modeling, and gene therapy.

References and further reading

1. Kim, J., Koo, B.-K. and Knoblich, J.A. (2020). Human organoids: model systems for human biology and medicine. Nature Reviews Molecular Cell Biology, (online) 21(10), pp.571–584. https://doi.org/10.1038/s41580-020-0259-3.
2. Zhao, Z. (2022). Organoids. Nature Reviews Methods Primers, (online) 2(1), pp.1–21. https://doi.org/10.1038/s43586-022-00174-y.

Acknowledgments

Produced from materials originally authored by Yingying Fu and Cheng Wang from Innovec Biotherapeutics; and Zhen Qi, Zhanguang Zuo, Spencer Chiang, An Ouyang, Glory Gao, Shuge Guan, Jessie Chen, and Rosanna Zhang from ACROBiosystems.

About ACROBiosystems

ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.

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