Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps

Brimble, Mark A.; Cheng, Pei-Hsin; Winston, Stephen M.; Reeves, Isaiah L.; Souquette, Aisha; Spence, Yunyu; Zhou, Junfang; Wang, Yong-Dong; Morton, Christopher L.; Valentine, Marcus B.; Thomas, Paul G.; Nathwani, Amit C.; Gray, John T.; Davidoff, Andrew M.

doi:10.1016/j.omtm.2022.01.008

Cited by 10 publications

(4 citation statements)

References 40 publications

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“…In the case of DNA impurities acquired from plasmids used for transfection, preferential packaging of plasmid DNA fragments containing the P5 promoter sequence has been reported. 33 These studies suggest that DNA impurities may be packaged into capsids in a single-stranded form. 9 Elucidating the form of host cell DNA packaged into capsids and preferential packaging is necessary even though single-stranded DNA may not have a risk similar to that of double-stranded DNA.…”

Section: Discussionmentioning

confidence: 99%

Quantitation of Residual Host Cell DNA in Recombinant Adeno-Associated Virus Using Droplet Digital Polymerase Chain Reaction

et al. 2023

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Recombinant adeno-associated virus (rAAV) is a viral vector commonly used in gene therapy. Residual host cell DNA is an impurity that has been associated with the risk of infection and oncogenicity. Thus, it needs to be monitored for quality control. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) method targeting 18S ribosomal RNA (rRNA) genes to quantitate residual host cell DNA. The copy number of the 18S rRNA gene was determined using two sets of primer pairs for 116- and 247-bp amplicons sharing the C-terminus. For conversion of the copy number of the 18S rRNA gene into the mass concentration of genomic DNA, the accurate copy number of 18S rRNA genes in HEK293 genomic DNA was determined by comparison with copy numbers of three reference genes ( EIF5B , DCK , and HBB ). Results showed that 88.6–97.9% of HEK293 genomic DNA spiked into rAAV preparations was recovered. The ddPCR-based assay was applied to rAAV preparations to quantitate residual host cell DNA as an impurity. Our findings indicate that the assay can be used for the quantitation and size distribution of residual host cell DNA in rAAV products.

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Section: Discussionmentioning

confidence: 99%

Quantitation of Residual Host Cell DNA in Recombinant Adeno-Associated Virus Using Droplet Digital Polymerase Chain Reaction

et al. 2023

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“…To date, approved rAAV vector-based gene therapies in humans are largely based on the monogenic replacement or upregulation of gene products known to the host. Even in this immunologically simpler scenario, rep gene sequence contaminants can find their way into virus preparations and contribute to immunogenicity [255]. For gene editing applications, the expression of Cas enzymes and their derivatives poses additional immunological challenges for human translation.…”

Section: Raav Vector-dependent Toxicity and Immune Responses In Humansmentioning

confidence: 99%

Advances in Recombinant Adeno-Associated Virus Vectors for Neurodegenerative Diseases

Li,

Vasan,

Kartono

et al. 2023

Biomedicines

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Recombinant adeno-associated virus (rAAV) vectors are gene therapy delivery tools that offer a promising platform for the treatment of neurodegenerative diseases. Keeping up with developments in this fast-moving area of research is a challenge. This review was thus written with the intention to introduce this field of study to those who are new to it and direct others who are struggling to stay abreast of the literature towards notable recent studies. In ten sections, we briefly highlight early milestones within this field and its first clinical success stories. We showcase current clinical trials, which focus on gene replacement, gene augmentation, or gene suppression strategies. Next, we discuss ongoing efforts to improve the tropism of rAAV vectors for brain applications and introduce pre-clinical research directed toward harnessing rAAV vectors for gene editing applications. Subsequently, we present common genetic elements coded by the single-stranded DNA of rAAV vectors, their so-called payloads. Our focus is on recent advances that are bound to increase treatment efficacies. As needed, we included studies outside the neurodegenerative disease field that showcased improved pre-clinical designs of all-in-one rAAV vectors for gene editing applications. Finally, we discuss risks associated with off-target effects and inadvertent immunogenicity that these technologies harbor as well as the mitigation strategies available to date to make their application safer.

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“…Some of the DNA process-related impurities in addition to interacting with foreign DNA recognition pathways that initiate innate immune and antiviral response may also be transcriptionally active and integrate into host cell DNA. 6 The role that various types and quantities of process-related impurities have in the toxicity profile of AAV gene therapy is poorly understood and is an area for further investigation.…”

mentioning

confidence: 99%

“…Regardless of the production method, the therapeutic product is a complex mixture of viral particles that contain the intended DNA expression construct as well as process-related impurities that consist of capsids that lack DNA (eg, empty capsids) and partially filled capsids. 4,6,[35][36][37][38]42 In addition to the intended DNA expression construct, the encapsulated DNA is a mixture of unintended DNA forms consisting of truncated expression construct, chimeric DNA that is a mixture of the DNA from the plasmids required for vector production, and the production cell line DNA as well as free fragments of DNA from the same sources. Other process-related impurities that are in the final product may also include proteins from the production cell line as well as reagents used in the purification process.…”

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confidence: 99%

An Overview of Nonclinical and Clinical Liver Toxicity Associated With AAV Gene Therapy

Whiteley

2023

Toxicol Pathol

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This article reviews the presentation given at the 2023 annual meeting of the Society of Toxicologic Pathology (STP) on liver toxicity observed with adeno-associated viral vector (AAV) gene therapy. After decades as a therapeutic modality largely confined to the academic research environment, gene therapy has emerged in recent years as a rapidly expanding therapeutic approach in the biopharmaceutical industry with AAV as the most commonly used viral vector for gene delivery. This interest in the field of gene therapy by industry has been enhanced by the recent success of approved therapies for curing genetic diseases such as ZOLGENSMA for spinal muscular atrophy and LUXTURNA for Leber congenital amaurosis. However, recently reported clinical and nonclinical toxicities highlight the challenges in safely developing AAV gene therapies that require high dose systemic administration. The presentation reviewed general attributes of AAV as a gene therapy vector, clinical and nonclinical liver toxicity associated with AAV gene therapy and the potential for a multimodal immune suppression strategy that may mitigate toxicities.

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Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps

Cited by 10 publications

References 40 publications

Quantitation of Residual Host Cell DNA in Recombinant Adeno-Associated Virus Using Droplet Digital Polymerase Chain Reaction

Quantitation of Residual Host Cell DNA in Recombinant Adeno-Associated Virus Using Droplet Digital Polymerase Chain Reaction

Advances in Recombinant Adeno-Associated Virus Vectors for Neurodegenerative Diseases

An Overview of Nonclinical and Clinical Liver Toxicity Associated With AAV Gene Therapy

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