Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors

Pekrun, Katja; Alencastro, Gustavo de; Luo, Qing; Li, Jun; Kim, Young‐Jin; Nygaard, Sean; Galivo, Feorillo; Zhang, Feijie; Ren, Shiyan; Tiffany, Matthew; Xu, Jianpeng; Hebrok, Matthias; Grompe, Markus; Kay, Mark A.

doi:10.1172/jci.insight.131610

Cited by 78 publications

(81 citation statements)

References 77 publications

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“…1A). 10 This 18 AAV capsid pool was used for initial studies as it has a low complexity and thus represents a powerful tool to optimize AAV library screens. The starting proportion of each of the barcodes is represented in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The method for generation of a pool consisting of a defined number of parental capsid AAVs is described in Ref. 10 Briefly, capsids from 18 AAV serotypes (AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV8, AAV9, AAV12, AAVporcine1, AAVporcine2, AAVbovine, AAVgoat1, AAVmouse1, AAVavian, AAVrh10, AAV-DJ, and AAV-LK03) were cloned into the BC library vector. Each AAV capsid contained a unique barcode sequence.…”

Section: Generation Of the 18 Aav Capsid Poolmentioning

confidence: 99%

“…The method for generation of a complex AAV capsid library is described in Ref. 10 Briefly, capsid sequences from eight AAV serotypes (AAV1, AAV2, AAV3B, AAV6, AAV8, AAV9, AAVporcine2, and AAVrh10), as well as from two shuffled variants AAV-DJ and AAV-LK03 that had been selected in previous screens, 1,2 were used as parental sequences for the shuffling reactions. After shuffling and amplification, fragments were ligated into the BC library vector (described in Ref.…”

Section: Generation Of 10 Aav Capsid Shuffled Librarymentioning

confidence: 99%

“…After shuffling and amplification, fragments were ligated into the BC library vector (described in Ref. 10 ). AAVs were purified and tittered as described above for the generation of the 18 AAV capsid pool.…”

Section: Generation Of 10 Aav Capsid Shuffled Librarymentioning

confidence: 99%

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Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing

et al. 2020

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Despite early successes using recombinant adeno-associated virus (rAAV) vectors in clinical gene therapy trials, limitations remain making additional advancements a necessity. Some of the challenges include variable levels of pre-existing neutralizing antibodies and poor transduction in specific target tissues and/or diseases. In addition, readministration of an rAAV vector is in general not possible due to the immune response against the capsid. Recombinant adeno-associated virus (AAV) vectors with novel capsids can be isolated in nature or developed through different directed evolution strategies. However, in most cases, the process of AAV selection is not well understood and new strategies are required to define the best parameters to develop more efficient and functional rAAV capsids. Therefore, the use of barcoding for AAV capsid libraries, which can be screened by high-throughput sequencing, provides a powerful tool to track AAV capsid evolution and potentially improve AAV capsid library screens. In this study, we examined how different parameters affect the screen of two different AAV libraries in two human cell types. We uncovered new and unexpected insights in how to maximize the likelihood of obtaining AAV variants with the desired properties. The major findings of the study are the following. (1) Inclusion of helper-virus for AAV replication can selectively propagate variants that can replicate to higher titers, but are not necessarily better at transduction. (2) Competition between AAVs with specific capsids can take place in cells that have been infected with different AAVs. (3) The use of low multiplicity of infections for infection results in more variation between screens and is not optimal at selecting the most desired capsids. (4) Using multiple rounds of selection can be counterproductive. We conclude that each of these parameters should be taken into consideration when screening AAV libraries for enhanced properties of interest.

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Section: Resultsmentioning

confidence: 99%

Section: Generation Of the 18 Aav Capsid Poolmentioning

confidence: 99%

Section: Generation Of 10 Aav Capsid Shuffled Librarymentioning

confidence: 99%

Section: Generation Of 10 Aav Capsid Shuffled Librarymentioning

confidence: 99%

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Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing

et al. 2020

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“…It has also been shown that AAV neutralizing antibodies do not have as much of an effect when inoculations are administered intramuscularly (Greig et al, 2016), which makes skeletal muscle tissue an easier target compared to the liver. Another approach could be through the use of synthetic capsids (Büning and Srivastava, 2019;Pekrun et al, 2019). Both rational design and directed evolution studies are being employed to generate synthetic capsids that improve AAV transduction to specific tissues and avoid the pre-existing neutralizing antibody responses.…”

Section: Current Limitations When Using Raav Vectorsmentioning

confidence: 99%

Promise and Progress of an HIV-1 Cure by Adeno-Associated Virus Vector Delivery of Anti-HIV-1 Biologics

Gardner

2020

Front. Cell. Infect. Microbiol.

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Despite the success of antiretroviral therapy (ART) at suppressing HIV-1 infection, a cure that eradicates all HIV-1-infected cells has been elusive. The latent viral reservoir remains intact in tissue compartments that are not readily targeted by the host immune response that could accelerate the rate of reservoir decline during ART. However, over the past decade, numerous broadly neutralizing antibodies (bNAbs) have been discovered and characterized. These bNAbs have also given rise to engineered antibody-like inhibitors that are just as or more potent than bNAbs themselves. The question remains whether bNAbs and HIV-1 inhibitors will be the effective "kill" to a shock-and-kill approach to eliminate the viral reservoir. Additional research over the past few years has sought to develop recombinant adeno-associated virus (rAAV) vectors to circumvent the need for continual administration of bNAbs and maintain persistent expression in a host. This review discusses the advancements made in using rAAV vectors for the delivery of HIV-1 bNAbs and inhibitors and the future of this technology in HIV-1 cure research. Numerous groups have demonstrated with great efficacy that rAAV vectors can successfully express protective concentrations of bNAbs and HIV-1 inhibitors. Yet, therapeutic concentrations, especially in non-human primate (NHP) models, are not routinely achieved. As new studies have been reported, more challenges have been identified for utilizing rAAV vectors, specifically how the host immune response limits the attainable concentrations of bNAbs and inhibitors. The next few years should provide improvements to rAAV vector delivery that will ultimately show whether they can be used for expressing bNAbs and HIV-1 inhibitors to eliminate the HIV-1 viral reservoir.

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Gene Therapy for Diabetes

Jimenez,

Bosch

2024

Textbook of Diabetes

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Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors

Cited by 78 publications

References 77 publications

Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing

Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing

Promise and Progress of an HIV-1 Cure by Adeno-Associated Virus Vector Delivery of Anti-HIV-1 Biologics

Gene Therapy for Diabetes

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