Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

Gao, Kai; Li, Mengxin; Zhong, Li; Su, Qin; Li, Jia; Li, Shaoyong; He, Ran; Zhang, Yu; Hendricks, Gregory M.; Wang, Junzhi; Gao, Guangping

doi:10.1038/mtm.2013.9

Cited by 109 publications

(99 citation statements)

References 30 publications

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“…These data tabulated in (Table 1) suggest that total capsid (cp) doses of at least 1 × 10 12 cp/kg may be necessary to surmount even low level of preexisting NAbs. Indeed, Mingozzi et al showed in a mouse model of anti-AAV NAbs that empty capsids can enhance FIX transgene expression in a dose-dependent manner that is also a function of NAb titer; however, excessive empty capsids also lead to decreased transgene expression, likely due to competition with vector at cellular binding sites [24] since the addition of empty capsids significantly decreases transduction in mice without NAbs [25]. Together, the human and mouse data suggest that reintroduction of empty viral capsids is a promising strategy for avoiding NAbs, especially at low titer levels.…”

Section: Overcoming Immune Responses To Aavmentioning

confidence: 99%

Obstacles and future of gene therapy for hemophilia

Arruda

Samelson‐Jones

2015

Expert Opinion on Orphan Drugs

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Introduction The recent success of early-phase clinical trials for adeno-associated viral (AAV) liver-directed gene therapy for hemophilia B (HB) demonstrates the potential for gene therapy, in the future, to succeed protein-based prophylaxis therapy for HB. Significant obstacles, however, need to be overcome prior to widespread adoption. The largest obstacles include immune responses to the AAV capsid including preexisting neutralizing antibodies (NAbs) and a delayed cellular immune response. Emerging evidence suggests that the latter is vector-dose dependent. Furthermore, the development and eradication of inhibitors remains a significant safety concern. Similarly, biological differences between Factor VIII and Factor IX (FIX) impose challenges to direct adoption of the successes for HB to hemophilia A (HA). Areas covered The advantages and limitations of the current strategies addressing these obstacles for gene therapy for HB and HA are discussed, as well as vector manufacturing issues relevant to widespread adoption. Alternative strategies including both ex-vivo and in-vivo lentiviral-based methods are discussed, though we focus on AAV-based approaches because of their recent clinical success and potential. Expert opinion Our opinion is that these obstacles can be overcome with current approaches, and AAV-based gene therapy for HB will likely translate into future clinical care. Innovative approaches are, however, likely needed to solve the current problems obstructing HA gene therapy.

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Section: Overcoming Immune Responses To Aavmentioning

confidence: 99%

Obstacles and future of gene therapy for hemophilia

Arruda

Samelson‐Jones

2015

Expert Opinion on Orphan Drugs

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“…Most of the negatively stained pseudo-vectors appeared to be empty because they displayed donut-like shapes of virions, and no significant difference was found among the three types of pseudo-vectors (Figure 3A) 15 . We also did not observe self-assembly intermediates, which should be smaller than the regular particles 20 .…”

Section: Resultsmentioning

confidence: 73%

“…Recently, Gao et al. demonstrated that pseudo-vectors suppressed transgene expression and contributed to hepatic transaminase elevation in mice 15 . Moreover, the pseudo-vectors cogenerated and separated from the bona fide AAV8 vectors, resulting in higher elevations of liver enzyme than the vector alone or mixed with a completely empty vector.…”

Section: Introductionmentioning

confidence: 99%

Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors

Wang

Dong

Pokiniewski

et al. 2017

Molecular Therapy - Methods & Clinical Development

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Adeno-associated virus (AAV) vectors have emerged as a safe and efficient gene therapy platform. One complication is that a significant amount of empty particles have always been generated as impurities during AAV vector production. However, the effects of such particles on AAV vector performance remain unclear. Here we systemically evaluated the biological properties of three types of “empty” AAV particles: syngeneic pseudo-vectors with partial AAV genomes derived from DNA of the corresponding full particles, allogeneic pseudo-vectors with partial genomes different from the corresponding full particles, and null pseudo-vectors with no DNA inside the capsids. The syngeneic particles in excess increased the corresponding full AAV vector transgene expression both in vivo and in vitro. However, such effects were not observed with null or allogeneic particles. The observed differences among these pseudo-AAV particles may be ascribed to the syngeneic pseudo-vector DNA facilitating the complementary DNA synthesis of the corresponding full AAV particles. Our study suggests that the DNA content in the pseudo-vectors plays a key role in dictating their effects on AAV transduction. The effects of residual “empty” particles should be adequately assessed when comparing AAV vector performance. The syngeneic AAV pseudo-vectors may be used to enhance the efficacy of gene therapy.

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“…Immunogenicity of AAVs may be exacerbated by empty capsid contamination in viral preparations 35,36 . The viral purification protocol (Steps 15-31) provided here reduces, but does not eliminate, empty capsids (Fig.…”

Section: Limitations Of the Methodsmentioning

confidence: 99%

Widespread and targeted gene expression by systemic AAV vectors: Production, purification, and administration

Challis

Kumar

Chan

et al. 2018

Preprint

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We recently developed novel AAV capsids for efficient and noninvasive gene transfer across the central and peripheral nervous systems. In this protocol, we describe how to produce and systemically administer AAV-PHP viruses to label and/or genetically manipulate cells in the mouse nervous system and organs including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression.The protocol spans eight days, excluding the time required to assess gene expression, and can be readily adopted by laboratories with standard molecular and cell culture capabilities. We provide guidelines for experimental design and choosing the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

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Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

Cited by 109 publications

References 30 publications

Obstacles and future of gene therapy for hemophilia

Obstacles and future of gene therapy for hemophilia

Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors

Widespread and targeted gene expression by systemic AAV vectors: Production, purification, and administration

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