Despite the widespread use of wild-type AAV viruses as delivery vectors in gene therapy, limitations still exist for tissue-specific vector expression. To overcome these limitations, Cyagen has developed AI-assisted high-efficiency screening technology to offer next generation AAV vectors for in vitro cell or in vivo animal model studies, ensuring quality AAV vectors with standardized viral titers that are purified and ready for preclinical research.
Taking neurodegenerative disease research as an example, the natural AAV9 serotype is severely limited in its ability to transduce the brain due to the blood-brain barrier. Traditional directed evolution and rational design methods yield a limited number of AAV9 variants and are time-consuming. Therefore, Cyagen utilizes AI technology to guide the design of high-performance and diverse sequence variants. After injection into mice, NGS sequencing is employed to track the results, leading to the selection of a large number of AAV9 variants with high specificity and expression in the central nervous system.
Utilizing our in-house developed AI technology and wet lab methods, we can provide novel AAV capsid variants that are safer, more convenient, and precisely target specific sites. Their specificity and transduction efficiency have been validated through multidimensional data analysis. To support research on neurological, ophthalmic, and other diseases, we are now offering free trials of various types of AAV capsid variants. We look forward to advancing research and drug development in relevant diseases. Please feel free to contact us at 800-921-8930 or email us at animal-service@cyagen.com.
Event Period: February 27, 2024 - May 31, 2024
Free Trial of AAV9 Capsid Variants
Free Trial of AAV2 Capsid Variants
| Group | Virus | Expression Capacity in Different Brain Regions | Trial Dosage of CAG-EGFP | Apply for Trial | |||||
|---|---|---|---|---|---|---|---|---|---|
| Hippocampus | Cortex | Corpus Callosum | Midbrain | Spinal Cord | Liver | ||||
| Wild-type Control | AAV9 | 1 | 1 | 1 | 1 | 1 | 1 | 1E12vg | |
| Positive Control | AAV9.phpeB | 13.2 | 17.3 | 12.8 | 20.2 | 18.7 | 0.16 | 1E12vg | |
Novel AAV9 Capsid Variant 1
|
PM167 | 18.5 | 19.2 | 16.4 | 23.2 | 24.4 | 0.25 | 1E12vg | |
| Novel AAV9 Capsid Variant 2
|
PM170 | 8.6 | 13.2 | 5.9 | 25.3 | 19.7 | 0.14 | 1E12vg | |
| Group | Virus | Retinal Back Layer Penetration Capability |
Whole-Eye Expression Capability |
Trial Dosage of CAG-EGFP |
Apply for Trial |
|---|---|---|---|---|---|
| Wild-type Control | AAV2-WT | 0 | 1 | 5E10vg | |
| Positive Control | AAV2.7M8 | 1 | 2 | 5E10vg | |
| Novel AAV9 Capsid Variant 1
|
PM077 | 3 | 10 | 5E10vg | |
| Novel AAV9 Capsid Variant 2
|
PM021 | 1.5 | 12.5 | 5E10vg | |
| Novel AAV9 Capsid Variant 3
|
PM054 | 10 | 15 | 5E10vg |
| Service | Specification | Titer | Timeline | Order |
|---|---|---|---|---|
| Adeno-Associated Virus (AAV) Packaging |
1×10¹² GC | ≥5×10¹² GC/ml | As Fast As 3 Weeks | |
| 2×10¹² GC | ≥5×10¹² GC/ml | |||
| 5×10¹² GC | ≥1×10¹³ GC/ml | |||
| 1×10¹³ GC | ≥1×10¹³ GC/ml | |||
| 2×10¹³ GC | ≥1×10¹³ GC/ml | |||
| Others | Others |
| Service | Specification | Titer | Timeline | Order |
|---|---|---|---|---|
| Adeno-Associated Virus (AAV) Packaging |
1×10¹² GC | ≥5×10¹² GC/ml | As Fast As 3 Weeks | |
| 2×10¹² GC | ≥5×10¹² GC/ml | |||
| 5×10¹² GC | ≥1×10¹³ GC/ml | |||
| 1×10¹³ GC | ≥1×10¹³ GC/ml | |||
| 2×10¹³ GC | ≥1×10¹³ GC/ml | |||
| Others | Others |
*Additionally, we can offer AAV viruses in other specifications, as well as packaging services for other virus types such as lentivirus and adenovirus. Please feel free to reach out to us at 800-921-8930 or email us at animal-service@cyagen.com for further inquiries.
Validation Data
Cyagen has developed an AI platform that integrates big data, cloud computing, machine learning, and other technologies to optimize the AAV9 capsid protein. This has generated a large number of candidate variants. The results show a high level of confidence in the predicted liver de-targeting data (Figure 1), with a PearsonR correlation coefficient as high as 0.884.
Figure 1. Predicted Liver Infection Efficiency
Top sequences selected from AI-predicted sequences were individually validated by tail vein injection in mice (5E11 vg/each) and examined after 21 days. In vivo imaging results (Figure 2) demonstrate that PM167 exhibits significantly better liver de-targeting than PHP.eB, while PM170 shows significantly better liver de-targeting than AAV9 wild-type (WT) and slightly higher than PHP.eB.
Figure 2. In Vivo Validation of Liver Infection Efficiency (In Vivo Imaging
- Luc)
After tail vein injection in mice (5E11 vg/each) and a 21-day incubation period, frozen section results (Figure 3) reveal that PM167 exhibits significantly lower green fluorescent protein signals in the liver compared to PHP.eB. PM170, on the other hand, shows slightly higher green fluorescent protein signals in the liver than PHP.eB but still significantly lower than AAV9 wild-type (WT).
Figure 3. In Vivo Validation of Liver Infection Efficiency (Frozen Section
EGFP)
Cyagen has developed an AI platform that integrates big data, cloud computing, machine learning, and other technologies to optimize the AAV9 capsid protein. This has generated a large number of candidate variants. The results show a high level of confidence in the predicted central nervous system targeting data (Figure 4), with a PearsonR correlation coefficient as high as 0.843.
Figure 4. Predicted Central Nervous System
Infection Efficiency
Top sequences selected from AI-predicted sequences were individually validated by tail vein injection in mice (5E11 vg/each) and examined after 21 days. In vivo imaging results (Figure 5) show a high accumulation of Top sequences in the brain. The Luc signal intensity in the brain expressed by PM167 is approximately two times that of PHP.eB, while the Luc signal intensity in the brain expressed by PM170 is approximately 1.5 times that of PHP.eB.
Figure 5. In Vivo Validation of Brain Infection Efficiency (In Vivo Imaging,
Luc)
To further investigate the distribution of Top sequences in different regions of the central nervous system, we conducted another examination in mice after tail vein injection (5E11 vg/each) and a 21-day incubation period. Frozen section results (Figure 6) demonstrate that PM167 exhibits significantly higher green fluorescent protein signals than PHP.eB in various brain regions (cortex, corpus callosum, hippocampus, midbrain) and the spinal cord. PM170 also shows higher green fluorescent protein signals than PHP.eB in various brain regions and the spinal cord, except for the corpus callosum.
Top sequences selected from AI-predicted sequences were pooled in equal ratios into a single test article and used to deliver barcoded transgene reporters respectively. We injected the mixed test article (5E12 vg total) into the cisterna magna of a non-human primate (♀, 3.6 kg). Following 16 days in-life, animal was sacrificed and tissues were processed for next generation sequencing (NGS) and and histology.
System Infection Efficiency (NGS)
System Infection Efficiency (Frozen Slice EGFP)
Using the Cyagen AI-AAV platform, we constructed a high-capacity mutant plasmid library, packaged a virus library, and performed NGS sequencing. We built a DualConvLSTM network to establish an AAV2 production prediction model. The model's credibility was validated on the test set, achieving a high correlation with Pearson=0.929 and Spearman=0.859 (Figure 9). Additionally, the AI-generated retinal targeting model showed a correlation of Pearson=0.874 and Spearman=0.871 on the test set (Figure 10).
We used the production model and the retinal targeting AI model to predict variants with high production and expression capabilities. We selected the top sequences and constructed RC mutant plasmids. These plasmids were separately packaged with wild-type AAV2 plasmids and 7M8 plasmids to produce Luciferase viruses. After virus packaging, purification, and QPCR titer testing, all three variants showed higher yields compared to AAV2 and AAV2.7M8. Specifically, PM054 had the highest yield, which was 3.48 times that of AAV2. PM021 and PM077 had yields 1.5 times and 2.01 times that of AAV2, respectively.
Figure 11. AI Predicted AAV2 Variant Virus Yields
The packaged Luciferase viruses were injected into the vitreous cavity of mice at a dosage of 3E+9 vg (viral genomes) per eye. After 3 weeks, Luciferase expression was detected using both in vivo imaging (Figure 12) and chemiluminescence assays. The in vivo imaging results showed that the signal intensity of Luciferase for all three variants was higher than that of AAV2 and AAV2.7M8.
For a more precise quantification, mice were euthanized, and their eyeballs were collected and homogenized for chemiluminescence detection. The PM054 variant exhibited the highest Luciferase expression level, which was 15 times that of AAV2. PM021 and PM077 had Luciferase expression levels of 12.5 times and 10 times that of AAV2, respectively.
Figure 12. In vivo Validation of Whole Eye Expression
Capability (In Vivo Imaging)
Figure 13. In vivo Validation of Whole Eye
Expression Capability (Chemiluminescence)
To further validate the in vivo infection efficiency of the variants and explore the cell types infected, EGFP viruses were packaged and injected into the vitreous cavity of mice (3E+9 vg per eye). After 3 weeks, the overall EGFP expression was detected through fundus fluorescence photography, and eyeball samples were collected for pathological examination. The fundus photography results (Figure 14) showed that the GFP signal of PM054 was the strongest, and the fluorescence signals of all three variants were significantly higher than those of AAV2 and AAV2.7M8.
DAPI staining was performed on frozen sections of eyeballs, and the results showed that the infection range of all three variants was greater than that of AAV2-WT and AAV2.7M8. In particular, PM021 and PM054 could infect almost the entire retinal area. In terms of the infection depth, AAV2-WT only infected the RGC layer cells, while AAV2.7M8 had some penetration capability, infecting a small number of optic nerve cells in the posterior retina. All three variants exhibited greater penetration capability than AAV2-WT and AAV2.7M8, infecting cells in various layers of the retina from RGC to PRC. Among them, PM054 had the best infection rate and expression in optic nerve cells, with an infectivity in the posterior retina approximately 10 times that of AAV2.7M8.
Platform Advantages
