Gene therapy is a promising technique for the treatment of a wide range of diseases at the genetic level, addressing the root cause of many subsequent disorders. Recent advancements have facilitated the development of more personalized and targeted treatments, marking a fundamental shift towards precision medicine.1
However, despite its potential, gene therapies face significant challenges, particularly in optimizing gene delivery methods for precise tissue targeting. A key obstacle is translating from preclinical to clinical models, especially in the context of viral vector optimization.2
Adeno-associated viruses (AAVs) are the primary vectors for gene delivery, known for their non-pathogenic nature and transgene delivery mechanisms that transfer genetic material into a wide range of cell types.
AAV vectors are widely used in the scientific community and are available through commercial suppliers, yet their capsids exhibit high variability, with different serotypes displaying different transduction efficiencies.3 Therefore, targeted gene therapies need a tailored vector to enhance the specificity and transgene delivery efficiency to target organs, tissues or cell types.
Existing AAV transgene delivery efficacy models introduce errors due to their simplicity or lack of alignment with human physiology.3 Selecting the wrong AAV parameters can lead to optimization errors and failure in clinical translation.4
In vitro models use engineered cell lines to display the desired aberrant expression profile. However, cell lines lack complex cell-cell interactions with a broad range of cell types, extracellular matrix interactions and signaling molecules within the culture. These interactions can all impact cellular structure, differentiation, and proliferation, altering AAV transgene delivery efficacy.
Despite these limitations, in vitro models are commonly adopted due to ease of use and scalability. In vivo models are more realistic representations of human physiology; however, they lack the human biomolecular features crucial for testing transduction efficacy. In addition, in vivo models have limited scalability and present ethical concerns, inhibiting their use in AAV capsid selection.
Human organoids are small three-dimensional structures cultivated from stem cells or tissue samples that replicate the architecture and function of specific organs and tissues.5 They offer an invaluable platform for the study of human biology, disease mechanisms and drug responses, providing a more physiologically relevant context than traditional cell cultures.
The capability to recapitulate complex tissue structures and functionalities, including cell-cell interactions and organization, combines the advantages of in vitro and in vivo methods.
By closely mimicking human tissue architecture and cellular diversity, organoids offer a more physiologically relevant environment to study AAV tropism, efficiency, transduction and transgene expression.6 In addition, organoids can replicate interactions that are not accessible using in vivo and in vitro methods.
Figure 1. Staining of Cerebral Organoids for Characterization. (a) Immunostaining of cerebral organoid at day 36 for neural stem cell marker NESTIN (red) and neuron TUJ1 (green) markers with nuclei visualized by a DAPI counterstain. Neural stemness remains near the borders of the organoid as shown by the NESTIN marker. (b) Day 119 of cerebral organoid shows presence of oligodendrocyte, shown by OLIG2 (pink) markers alongside neuron TUJ1 (green) markers. (c) Mature neuron marker (MAP2) dominates the borders as the organoid reaches its maintenance stage. (d, e) H&E stain of a 32-day-old organoid section reveals rosette-like structures within the 3D organoid structure. Image Credit: ACROBiosystems
In the study discussed in this article, a methodology to generate multiple organ-specific organoid models derived from human induced pluripotent stem cell (iPSC) sources is outlined. Each model is characterized and utilized to evaluate several AAV serotypes regarding transgene delivery efficacy.
The differences in AAV delivery efficacy between wild-type (WT) and manufactured serotypes are highlighted. A scalable methodology for selecting AAV serotypes between each organoid type is also presented to help select AAV vectors for more effective gene therapy interventions.
Development of organoid models
Organoids were cultured from healthy donor iPSC sources and differentiated using the human iPSC-derived Cerebral Organoid Differentiation Kit, human iPSC-derived Cardiac Organoid Differentiation Kit, and human iPSC-derived Retinal Organoid Differentiation Kit. Each organoid model was cultured following the specified protocols.
Cerebral organoids were cultured for 119 days, exhibiting cerebral-like structures with neurons, astrocytes, and oligodendrocytes, confirmed using their respective markers.
On day 36, organoid margins tested positive for the neural stem cell marker NESTIN, with high expression levels of the neuronal cell marker TUJ1, as shown in Figure 1a. By day 119, higher expression levels of MAP2 indicated the presence of mature neurons, while OLIG2 markers confirmed oligodendrocytes, as shown in Figures 1b and 1c.
Cerebral organoids at days 32 and 46 also exhibited rosette-like structures, indicative of neuronal or ependymal cell differentiation throughout the organoid, as shown in Figures 1d and 1e.
After reaching the maintenance stage (11 days after embryoid body formation), a preliminary evaluation of cardiac organoids was conducted. Contraction activity was visibly observed via light microscopy upon reaching maturity.
The cardiac organoid model also tested positive for the cardiomyocyte marker cardiac Troponin (cTnT), confirming the presence of cardiomyocytes throughout the organoid body, as shown in Figure 2.
Figure 2. Immunostaining of Cardiac Organoids. (a) Magnified view (20x) of cardiac organoids with cardiomyocytes were visualized using the cTnT marker with nuclei visualized by DAPI. (b) Presence of cardiomyocytes throughout the entire organoid can be observed. Image Credit: ACROBiosystems
Figure 3. Bulk RNA Sequencing of Cerebral Organoids. Various gene markers were monitored for the presence of various cell types and subtypes throughout the growth and maturation of generated cerebral organoids. Image Credit: ACROBiosystems
Development of organoid models
RNA sequencing for known gene markers
Cerebral organoid composition was evaluated using bulk RNA sequencing for each organoid model, as shown in Figure 3. Gene expression data was recorded for cerebral organoids on day 13, 44 and 100. Immature cell markers such as NES and PAX6 reduced as the organoids increased, showing maturation and differentiation of neural stem cells.
Likewise, there was an increase in MAP2, DLG4/PSD95 and SYP markers for mature cells. Cell diversity increased, evidenced by positive markers revealing an increase in expression of astrocytes, oligodendrocytes, and other neuron subclass markers.
Cardiac organoid function
Cardiac organoids were studied separately, as contraction activity was visually observed, closely mimicking the diastole and systole phases of cardiac conduction.
A silicon probe with multichannel reading capabilities was introduced to the organoid to capture impulse generation and conduction by detecting transient voltage changes during each pulse, as shown in Figure 4.
The electrical waveform recorded by the probe closely resembled the ventricular complex and T wave of a normal electrocardiogram.
Figure 4. Transient Voltage Potential across Cardiac Organoid. Silicon probes were placed across a cardiac organoid to measure the impulse generation and propagation causing the contraction activity. Each contraction lasts around 449 ms and can be continuously observed. Image Credit: ACROBiosystems
AAV transduction into human organoid models
To validate organoids as a model for AAV capsid selection, several manufactured serotypes were selected to compare against WT AAV2 and AAV5.
Each AAV variant carried a transgene encoding green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. This enabled the fluorescent expression to be assessed following in vitro delivery.
Cerebral organoids, matured for 101 days, were transferred to an organoid medium containing 1 x 1011 vg per well. Each well contained two organoids for viral transduction and were analyzed by fluorescent microscopy after 124 hours, as shown in Figures 5d, 5e and 5f.
At 11 days of growth, cardiac organoids were separately transferred to the organoid medium containing 1 x 1011 vg of AAVs per well with two organoids per well, as shown in Figures 5a, 5b and 5c. After 95 hours, the cardiac organoids were analyzed.
IVB-1 and IVB-2 were utilized for both AAV5 WT and custom serotypes. The majority of transduced cells were expected to be closer to the organoid margins as AAVs were placed into the organoid medium. This was confirmed for both organoids.
Transgene efficiency of the IVB-2 serotype was highly amenable in infecting cerebral organoids, indicating a high GFP intensity across the organoid, in contrast to the relatively limited GFP intensity of IVB2 in cardiac organoids.
Additionally, since IVB-1 and IVB-2 are related to AAV5-WT, the increased infectiousness towards cerebral organoids aligns with the effectiveness of AAV5 for treating neurological disorders, as reported in literature.7
Quantitative evaluation of retinal organoids
To evaluate transgene delivery efficiency, 185-day-old retinal organoids were loaded with AAV2-WT and three different serotypes: IVT-9, IVT-18 and IVT-21. For each organoid, 1 x 1010 vg of each AAV serotype was placed into the organoid medium. GFP expression was evaluated by flow cytometry after 7 days.
IVT21 displayed the greatest retinal organoid transduction efficiency (73.19 %), whereas AAV2-WT only displayed 34.40 %. The remaining serotypes exhibited 66.90 % and 46.96 %.
This evaluation provided a rapid quantitative methodology for screening and selection of AAV serotypes generated in parallel for transgene delivery optimization.
Figure 5. Fluorescent microscopy of organoids after infection by several AAV serotypes. (a,b,c) Cardiac organoids grown for 11 days were infected with (a) AAV5-WT, (b) IVB-1, (c) IVB-2 were individually placed into a well plate. Transgene delivery efficacy was visualized by fluorescent intensity from GFP transgene expression. (d,e,f) Cerebral organoids grown for 101 days were infected with (d) AAV5-WT, (e) IVB-1, and (c) IVB-2. Comparatively, the IVB-2 subtype showed higher infectiousness and transgene delivery towards cerebral organoids rather than cardiac, elucidating varying efficacy between organ types. Image Credit: ACROBiosystems
Conclusions
AAVs play a central role in gene therapy, serving as key vectors for gene delivery. However, selecting the optimal AAV serotype is challenging due to variability in transduction efficiency.
Complex cell-cell interactions and the cellular microenvironment are critical factors that standard in vitro screening methods may not fully capture. Additionally, non-human models lack human-specific characteristics, which can impact AAV efficacy.
This study presents a scalable method for characterizing and screening AAV serotypes to aid in viral vector selection for drug discovery. A range of known cell markers was used to assess organoid growth and cellular composition, confirming that the organoids accurately mimicked organ structure and function.
Multiple types of organoids, including cardiac, cerebral, and retinal models, were used as preclinical platforms for AAV selection across different organ systems. Differences between manufactured AAV serotypes and WT AAVs were evaluated based on GFP expression, with quantification performed through cell counting of GFP-expressing cells.
Organoids are anticipated to play a significant role in gene therapy development, particularly AAV selection, helping to address the gap between discovery and clinical translation.
Figure 6. Flow cytometry for transgene delivery efficacy in retinal organoids. Quantitative evaluation of transgene efficacy was performed by counting the number of cells expressing GFP using retinal organoids grown for 185 days. AAVs were incubated for 7 days with the organoids. (a) AAV2-WT, (b) IVT18, (c) IVT9, and (d) IVT21 resulted in a different level of transduction, with IVT21 performing the best with 73.19% of parent cells expressing the AAV transgene. Image Credit: ACROBiosystems
Acknowledgments
Produced from materials originally authored by Zhen Qi, Yingying Fu, Spencer Chiang, Wei Li, Huihui Han, Zhanguang Zuo, Shuge Guan, Jessie Chen, Rosanna Zhang and Cheng Wang from ACROBiosystems.
References and further reading
- Kohn, D.B., Chen, Y.Y. and Spencer, M.J. (2023). Successes and Challenges in Clinical Gene Therapy. Gene Therapy, [online] 30, pp.1–9. https://doi.org/10.1038/s41434-023-00390-5.
- Jiang, Z. and Dalby, P.A. (2023). Challenges in scaling up AAV-based gene therapy manufacturing. Trends in Biotechnology, 41(10), pp.1268–1281. https://doi.org/10.1016/j.tibtech.2023.04.002.
- Au, H.K.E., Isalan, M. and Mielcarek, M. (2022). Gene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings. Frontiers in Medicine, 8. doi:https://doi.org/10.3389/fmed.2021.809118.
- Liguore, W.A., et al. (2019). AAV-PHP.B Administration Results in a Differential Pattern of CNS Biodistribution in Non-human Primates Compared with Mice. Molecular Therapy, 27(11), pp.2018–2037. https://doi.org/10.1016/j.ymthe.2019.07.017.
- Pașca, S.P. (2018). The rise of three-dimensional human brain cultures. Nature, 553(7689), pp.437–445. https://doi.org/10.1038/nature25032.
- Depla, J.A., et al. (2020). Cerebral Organoids: A Human Model for AAV Capsid Selection and Therapeutic Transgene Efficacy in the Brain. Molecular Therapy - Methods & Clinical Development, 18, pp.167–175. https://doi.org/10.1016/j.omtm.2020.05.028.
- Tardieu, M., et al. (2017). Intracerebral gene therapy in children with mucopolysaccharidosis type IIIB syndrome: an uncontrolled phase 1/2 clinical trial. Lancet Neurology, 16(9), pp.712–720. https://doi.org/10.1016/s1474-4422(17)30169-2.
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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