Finding DNA Tags in AAV Stacks


In the mid-1990s, Hiroyuki Nakai, now an adeno-associated virus (AAV) researcher at the Oregon Health and Science University, moved to the United States. Nakai quickly became fascinated with the biology of small viruses and their potential as gene delivery vehicles, and AAV and its applications in gene therapy soon became his primary focus. “It’s a simple virus. If you change one amino acid, you can totally change the phenotype and can substantially enhance the infectivity at targeted tissues without infecting other nontargeted tissues,” Nakai explained. “As a scientist, this is really interesting to understand.”

Researchers find the potential of AAV vectors in gene therapy exciting for several reasons. For one, they exhibit reduced toxicity and immunogenicity when compared to other viral vectors, such as adenoviruses and retroviruses. Additionally, the highly customizable nature of the AAV capsid, the protein shell that encloses the viral single-stranded DNA and affects the vector’s affinity for cells and tissues, allows scientists to design AAV vectors that more efficiently deliver therapeutic genes to targeted organs.  

Although scientists have been testing AAV gene therapy applications for more than two decades, conventional selection methods are cumbersome. Scientists have to deal with inter-animal and inter-experimental variations when comparing the results of independent AAV screens. Also, when conducting studies in a single cell type or organ, they cannot tell whether AAV variants transduced more than one tissue, masking a lack of specificity that would potentially make them unsuitable for clinical use. Lastly, to validate a broad panel of candidate AAV vectors, scientists have to use a large number of laboratory animals, which imposes ethical constraints.    

A faster and broader AAV vector screening

Around 2010, Nakai conceptualized a new technology that helped solve these problems: the AAV Barcode-Seq approach.1 Motivated by the reduction in cost of next generation sequencing (NGS), Nakai wanted to apply this technology to biologically characterize multiple AAV in a high-throughput manner. 

It’s a cost effective, ethically better, time effective method to study a lot of different AAV at the same time.

-Leszek Lisowski, Children’s Medical Research Institute University of Sydney.

Nakai’s idea was to add small, unique DNA sequences to the viral genomes. Just like barcodes on products at the grocery store, these DNA sequences add unique tags to each AAV. In their AAV screening experiments, which typically involved a mixture of different AAV, the team did the amplification and sequencing of the barcodes to identify individual vectors. They also assessed the presence of specific AAV in different tissues. By following a series of common molecular biology steps, the researchers used the AAV Barcode-Seq technology to compare the relative transduction efficiencies of 12 different AAV strains in many different tissues, using as few as three mice. They also used the method to create AAV serotype 9 (AAV9) mutants and demonstrated how specific amino acid signatures in the capsid influence the behavior of a vector, including its tissue affinity, blood clearance, and antibody-mediated neutralization.

While this proof-of-concept study introduced the technology of DNA barcoding combined with NGS to the AAV field, it took researchers a while to appreciate the utility of the method after it was first made public, according to Nakai. That has changed now. “Many people in our field have started using our technology, and they have developed their own method by modifying our method,” Nakai said. 

Taking it to the next level

Nakai’s team made some of the first improvements to the technology by enabling AAV to be tracked at the RNA level.2 To do this, his team designed an AAV library composed of 25 recombinant AAV serotype 2 (AAV2) viral clones and placed the DNA barcode downstream of a promoter sequence in the AAV to ensure expression of the barcode in the RNA molecule. By transducing cells with the library, they confirmed that the barcode sequence appeared in the transcript, thus opening up the method to determine transgene expression.  

The technology’s ability to track AAV vectors at both the DNA and RNA levels piqued the interest of scientists like Leszek Lisowski, a molecular biologist and geneticist at the Children’s Medical Research Institute at the University of Sydney, who wanted to test the method for in vitro, in vivo, and ex vivo applications. “It’s a cost effective, ethically better, time effective method to study a lot of different AAV at the same time,” Lisowski said.

     Illustration showing DNA barcoding with next-generation sequencing

(1) To track the expression of a specific AAV genome in a pool, researchers add a unique pair of small barcodes consisting of a few nucleotides to the AAV sequence. Each viral genome also contains two flanking inverted terminal repeats (ITR) that enclose the following genes: a rep gene, which is required for viral genome replication and packaging; a cap gene, which is required for the expression of capsid proteins; and a polyadenylation signal (pA), which ensures proper processing and stability of the mRNA product.

(2) Scientists then mix the different barcoded AAV to form an AAV pool or library.

(3) Researchers inject the AAV mixture into a living model, such as a mouse. The different AAV will transduce, or infect, the animals’ tissues.

(4) Scientists collect the tissues of interest and extract DNA from the different cells.

(5) Viral DNA from the different AAV is amplified and prepared for NGS.

(6) Researchers analyze NGS data to study patterns of viral DNA expression, which may identify AAV that have a trophism for certain tissues, and may thus serve as better gene delivery vehicles when targeting that specific tissue. 

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He and his team selected 30 previously published AAV variants, a mixture of natural and bioengineered vectors, and assembled them into an AAV test kit to test the technology’s ability to track the vectors in different experimental conditions.3  Each AAV carried an enhanced green fluorescent protein (eGFP) transgene cassette alongside a unique six-nucleotide long barcode. Using this approach, the team confirmed previous findings that AAV vectors efficiently transduced specific tissues, such as the mouse brain and human hepatocytes, and uncovered a novel variant that effectively transduced ex vivo primary human fibroblasts.

Although the idea of adding a tag to track an AAV vector seems simple, the selection of this small DNA sequence and its placement within the viral genome needs to be carefully thought out for a specific use, according to Lisowski. “We may affect the stability of the RNA by adding a barcode to a very simple gene array. If it does, that means that something expressed with that barcode would get destroyed faster,” he explained. “When measuring the expression of the transgene, it will look like it was never there and that the vector didn’t work, but [it] worked. It is just that the RNA got destroyed. So, all barcodes have to be validated.”

The ability to compare different AAV using the AAV Barcode-Seq method is also useful when working with nonhuman primates. AAV screening studies in nonhuman primates, such as rhesus macaques, are often limited by the high variability in the results that partially stems from the animals’ more distinct genetic backgrounds and potential pre-existing immunity to some of the AAV. Since nonhuman primates are biologically closer to humans than mice, scientists are interested in testing AAV vectors in these models to identify the AAV variants with promising gene therapy applications in humans. 

This AAV barcode technology and other types of high-throughput technologies we and other labs are working on can transform the gene therapy field substantially in the next 10 years or so.

-Hiroyuki Nakai, Oregon Health and Science University.

Interested in probing a distinct panel of AAV vectors that could deliver anti-HIV therapies directly to T cells, Daniel Stone, a gene therapist in Keith Jerome’s laboratory at the Fred Hutchinson Cancer Center, collaborated with other researchers to optimize the process of AAV selection in nonhuman primates using the AAV Barcode-Seq method.4  “We wanted to identify some AAV serotypes that would be optimal for targeting either CD4+ T cells or muscle and liver as sites for expression of broadly neutralizing antibodies,” Stone said. “We decided to look at several serotypes at the same time that would potentially be able to target all those different populations.”

The team used six-nucleotide long barcodes to identify each AAV vector in a panel consisting of naturally occurring and capsid-engineered vectors. Using the AAV Barcode-Seq method, the researchers compared the performance of the AAV directly, minimizing the experimental variables that often hindered the assessment in such studies. “There are techniques available out there that enable you to identify something that would suit your particular indication, and you can do this on a smaller scale,” Stone said.

In Nakai’s lab, he and his team are now applying the technology to investigate diseases, such as some inherited chronic kidney diseases, which have a well understood genetic basis and a pressing need for novel therapies. 

Unlike other organs, AAV-mediated gene delivery to the kidney is characterized by highly variable transduction efficiency.5 In an attempt to identify novel AAV vectors that could potentially be used for renal gene therapy, Nakai and his team compared more than 45 AAV variants using AAV Barcode-Seq and identified six that showed enhanced kidney transduction and limited off-target transduction in mice when administered more locally.6 When administered systemically to a mouse model of chronic kidney disease, the best performing variant showed poor transduction in the kidneys. Although the finding was contrary to their expectations, it revealed the importance of the context of selection (healthy versus disease) when evaluating the performance of AAV candidates for renal gene therapy.

The future of AAV Barcode-Seq technologies

Even though AAV Barcode-Seq provides a platform for high-throughput screening of AAV vectors, the method has limitations. As researchers need to mix all vectors in the same pool, some raised concerns about AAV competition, Nakai recalled. To enter a cell, an AAV must bind to a receptor on the cell surface. If another AAV happens to bind to that receptor first, it could interfere with the entry of the first AAV. Although this is possible, evidence of AAV competition is still lacking, according to Lisowski. “We always talked about it as a theoretical possibility, but we’ve never seen it,” he said. 

According to Lisowski, a major drawback of the method is that researchers cannot tell which cell types within a tissue are being transduced. “This can be done when you study one vector at a time,” he explained. In this context, scientists like Lisowski are using AAV Barcode-Seq technologies to narrow down the selection of AAV vectors and identify the most promising ones, which are then studied individually.

Despite the limitations, Nakai stands by these technologies. “There are a lot [of] future promises in terms of developing disease specific or cell specific AAV that can be delivered at a much lower dose than we are now using,” Nakai said. “This AAV barcode technology and other types of high-throughput technologies we and other labs are working on can transform the gene therapy field substantially in the next 10 years or so.”

Hiroyuki Nakai is one of the founders of Capsigen Inc., where he acts as the chief science officer. He also receives royalties for AAV-related technologies licensed by Takara Bio Inc.

References

  1. Adachi K, et al. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing. Nat Commun. 2014;5:3075. 
  2. Adachi K, et al. 288. Development of a universal AAV Barcode-Seq system expressing RNA barcodes. Mol Ther. 2014;22:S111.
  3. Westhaus A, et al. High-throughput in vitro, ex vivo, and in vivo screen of adeno-associated virus vectors based on physical and functional transduction. Hum Gene Ther. 2020;31(9-10):575-589. 
  4. Stone D, et al. A multiplexed barcode approach to simultaneously evaluate gene delivery by adeno-associated virus capsid variants in nonhuman primates. Hepatol Commun. 2023;7(2):e0009. 
  5. Peek JL, Wilson MH. Cell and gene therapy for kidney disease. Nat Rev Nephrol. 2023;19(7):451-462. 
  6. Furusho T, et al. Enhancing gene transfer to renal tubules and podocytes by context-dependent selection of AAV capsids. Preprint. bioRxiv. 2023

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