Synonymous mutations in AAV Rep enhance genome packaging in a library selection
Curation statements for this article:-
Curated by eLife
eLife Assessment
Engineering of adeno-associated virus (AAV) replication proteins may provide new insights into Parvoviral replication. The authors created a useful collection of Rep protein variants with changes that alter the amino acid sequence, but these did not lead to clear improvements in how the virus worked. Instead, their screen showed that changes that do not alter the protein ("synonymous" mutations) and changes to the promoter were more common. As it stands the results are incomplete due to potential issues with the screening design. We encourage a more complete characterization, which may enhance the translational potential of the approach.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Abstract
When producing Adeno-Associated Virus (AAV) gene therapies, a significant fraction of capsids can lack the desired DNA cargo. In AAV, Rep proteins mediate DNA packaging and virus assembly, suggesting that changes in Rep activity, expression, or DNA binding might affect genome packaging. To understand how mutations in the Rep gene affect activity, we selected a library of Rep mutants for their ability to produce active virions. By sequencing the Rep gene following the purification of viruses that package AAV genomes, we identified Rep mutants having non-synonymous mutations with a range of cellular activities. Surprisingly, synonymous mutations within the p19 promoter were enriched to the greatest extent, increasing in abundance by 102 to 104 fold. When the most highly enriched mutant was used to package a synthetic DNA cargo into the AAV capsid, the packaging efficiency could not be differentiated from native Rep. These findings suggest that these synonymous mutations enhance AAV genome packaging into capsids by affecting Rep-DNA interactions. They also suggest that silent sequence changes in the DNA cargo packaged by Rep can be used to tune packaging DNA packaging efficiency.
Article activity feed
-
-
-
eLife Assessment
Engineering of adeno-associated virus (AAV) replication proteins may provide new insights into Parvoviral replication. The authors created a useful collection of Rep protein variants with changes that alter the amino acid sequence, but these did not lead to clear improvements in how the virus worked. Instead, their screen showed that changes that do not alter the protein ("synonymous" mutations) and changes to the promoter were more common. As it stands the results are incomplete due to potential issues with the screening design. We encourage a more complete characterization, which may enhance the translational potential of the approach.
-
Reviewer #1 (Public review):
Engineering of AAV replication proteins may provide new insights into Parvoviral replication and potentially enable improved recombinant AAV vector yield when incorporated into the manufacturing process. Silberg and colleagues report an AAV Rep library, that is an interesting and powerful approach, however, the screening design and subsequent experiments lack rigor and ultimately the results are premature. Overall, the manuscript does not accurately describe state-of-the-art in the field and has significant shortcomings with experimental design/data analysis. Key concerns are noted below:
The high enrichment of P19 variants in the library was likely an artifact of the fact they only transfected 20 ng of RepCap into their 5-plate preps. When such little Rep is provided, any boost in Rep expression levels will …
Reviewer #1 (Public review):
Engineering of AAV replication proteins may provide new insights into Parvoviral replication and potentially enable improved recombinant AAV vector yield when incorporated into the manufacturing process. Silberg and colleagues report an AAV Rep library, that is an interesting and powerful approach, however, the screening design and subsequent experiments lack rigor and ultimately the results are premature. Overall, the manuscript does not accurately describe state-of-the-art in the field and has significant shortcomings with experimental design/data analysis. Key concerns are noted below:
The high enrichment of P19 variants in the library was likely an artifact of the fact they only transfected 20 ng of RepCap into their 5-plate preps. When such little Rep is provided, any boost in Rep expression levels will have a major on yield. When more RepCap is provided, 10 ug in their later evaluation, small changes in Rep expression are unlikely to have major impacts on yield. A more effective strategy would have been to transfect a normal amount of DNA and then utilize serial passaging through infectious cycling to account for cross packaging.
Introduction:
- There are 7 FDA approved AAV gene therapies.
- The description of "shuffling" when citing Mietsczh et al is misapplied. The cited paper discusses rationally designed hybrids.
- The graphic represents a hybrid capsid, but the focus is rep. As such, this should be depicted differently.Figures 1 and 2 are validation of previously published findings and general optimization of the experimental conditions. These do not provide the reader any new insight or information.
In Figure 3: The experimental approach is limited. It is unclear how the subpooling of different conditions was performed. As mentioned above, their library transfection strategy will significantly bias the results. The enriched variants have not been evaluated - specifically, the enriched non-synonymous mutations have not been shown to yield higher titers when tested individually.
In Figure 4: The claim is made that "several synonymous mutations within the p19 promoter region increase Rep DNA packaging activity." However, Figure 4c does not show statistically significant differences in support of this claim. Additional supporting data is needed. Further, Authors state that the synonymous mutations are near the P19 promoter. However, looking at the sequence shown in figure 4, their annotation of the P19 promoter is not correct and the mutations are actually within the P19 promoter. Relatedly, the authors note that mutations enriched in the p19 region include additional tetranucleotide repeats. No synthetic variants with additional GCTCs have been generated to test this hypothesis. Further, these results would benefit from a Western blot and transcript analysis to confirm Rep52/40, expression levels of constructs. -
Reviewer #2 (Public review):
In the present study the authors have investigated the effects of mutations on Rep protein ability to package DNA within the gene therapy vector, AAV. A detailed investigation of Rep mutants selected from a library has been probed for their ability to produce active virions. While the concept is interesting the outcome effects are very limited.
The major issue is the lack of immediate applicability and relevance in the vector production pipeline for AAV. The authors have found that with the synthetic GFP transgene cargo, mutations of the p19 promoter did not lead to enhanced AAV vector packing. Thus the data is quite preliminary and a complete characterization may be necessary to further enhance the translational potential of the approach.
-
Reviewer #3 (Public review):
While the AAV capsid has long been the target of protein engineering, its Rep proteins have been comparatively less studied. Since Rep plays a variety of roles for genome replication and virion packaging, gaining a deeper mechanistic understanding of their function and/or engineering versions that enable higher packaging productivity would be of interest to the field. This study generates a library of non-synonymous mutations in AAV2 rep (intended to cover all 19 aa changes at all positions, and coming close), packaged an AAV with AAV9 capsid, and sequenced the results to assess which amino acid changes resulted in an enrichment/depletion of genomes containing that variant rep. They found that proline substitutions are disruptive, well known from protein mutagenesis studies. The most significant enrichment …
Reviewer #3 (Public review):
While the AAV capsid has long been the target of protein engineering, its Rep proteins have been comparatively less studied. Since Rep plays a variety of roles for genome replication and virion packaging, gaining a deeper mechanistic understanding of their function and/or engineering versions that enable higher packaging productivity would be of interest to the field. This study generates a library of non-synonymous mutations in AAV2 rep (intended to cover all 19 aa changes at all positions, and coming close), packaged an AAV with AAV9 capsid, and sequenced the results to assess which amino acid changes resulted in an enrichment/depletion of genomes containing that variant rep. They found that proline substitutions are disruptive, well known from protein mutagenesis studies. The most significant enrichment sfound, however, were a set of synonymous mutations (unintended members of the library, as the library was designed to contain non-synonymous mutations) that lie within the p19 promoter. However, attempts to package recombinant vector using these individual rep variants in the AAV helper construct did not increase viral titer.
A previous study conducted analogous mutagenesis on Rep: Jain et al., "Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production" eLife 2024 (cited here as reference 19). It is not clear that this current study is a significant advance relative to the prior, quite comprehensive study. Both generated a library of non-synonymous mutations and observed fitness effects on Rep. Because this study sequenced the full rep, rather than barcodes associated with each rep variant, it found the enrichment in the synonymous mutations. However, these should ideally advance a basic understanding of Rep biology and/or result in better AAV production, but they did neither. It is speculated in the Discussion that the mutations generated additional GCTC motifs in p19, elements that mediate protein-DNA interactions. However, the role of GCTC motifs is speculative, and no transcriptional analysis is conducted. Furthermore, as discussed above, the mutations do not result in higher viral titers. Perhaps there's a transcriptional effect at the much lower copy numbers of vector genome present during library selection vs. rAAV packaging. They also found stop codons in Rep domains thought to be required for viral packaging, but functional studies confirming the screening findings are not conducted. As a result, the biological or technical relevance of the findings are extremely unclear, and thus the impact is relatively low.
The description of herring DNA co-transfection and cross-packaging (which is a well-known pitfall) are somewhat technical and arguably don't merit too much main manuscript attention.
-
-