Rational engineering of a functional CpG-free ITR for AAV gene therapy

Pan, Xiufang; Yue, Yongping; Boftsi, Maria; Wasala, Lakmini P.; Tran, Ngoc Tam L.; Zhang, Keqing; Pintel, David J.; Tai, Phillip W. L.; Duan, Dongsheng

doi:10.1038/s41434-021-00296-0

Cited by 30 publications

(19 citation statements)

References 87 publications

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“…As a result, the AAV particles containing CpG-free ITR were revealed to have a threefold reduction in capsid yield and the same percentage of empty capsids compared to wild-type viral vectors. Transduction of the AAV vectors carrying a microdystrophin expression cassette in vivo demonstrated no difference between CpG-free and wild-type ITRs (54). Therefore, although AAV vectors with CpG-depleted transgene cassettes were observed to improve the efficiency of transduction in different ways (39,55), CpG motif depletion in ITRs had no such effect.…”

Section: Aav Itr Modificationsmentioning

confidence: 90%

“…Mutagenesis in the trs flanking regions has similarly been observed to reduce Rep nicking, but only by 20-50% (44); 4-the deletion of the BB' and CC' regions reduces viral productivity by 75%, meanwhile, provides an increased level of transgene expression in vitro and in vivo, reaching up to a 6.6-fold increase in some cases, compared to wild-type ITRs (47); 5-substitution of the entire D region of the 5 ITR by a non-AAV substitute sequence containing transcription factor binding sites (TF BS) led to increased transduction efficiency, possibly due to increased transgene expression (110). Pink color indicates the sequence containing TF BS 6-CpG depleted ITRs are stable in bacterial passaging, thus facilitating AAV production (54). Red markers used for a schematic depiction of guanine and cytosine substitutions to adenine and thimine.…”

Section: Aav Itr Modificationsmentioning

confidence: 99%

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AAV- based vector improvements unrelated to capsid protein modification

Shitik

Shalik²,

Yudkin³

2023

Front. Med.

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Recombinant adeno-associated virus (rAAV) is the leading platform for delivering genetic constructs in vivo. To date, three AAV-based gene therapeutic agents have been approved by the FDA and are used in clinical practice. Despite the distinct advantages of gene therapy development, it is clear that AAV vectors need to be improved. Enhancements in viral vectors are mainly associated with capsid protein modifications. However, there are other structures that significantly affect the AAV life cycle and transduction. The Rep proteins, in combination with inverted terminal repeats (ITRs), determine viral genome replication, encapsidation, etc. Moreover, transgene cassette expression in recombinant variants is directly related to AAV production and transduction efficiency. This review discusses the ways to improve AAV vectors by modifying ITRs, a transgene cassette, and the Rep proteins.

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Section: Aav Itr Modificationsmentioning

confidence: 90%

Section: Aav Itr Modificationsmentioning

confidence: 99%

AAV- based vector improvements unrelated to capsid protein modification

Shitik

Shalik²,

Yudkin³

2023

Front. Med.

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“… 74 More recently, this technique was used to produce a CpG-free ITR that resulted in a therapeutic micro-dystrophin vector when tested in mice. 75 The authors speculate that the vector is less immunogenic, but further studies are needed to confirm the potential immunological advantage. 75 dsRNA, formed when the AAV ITRs have promoter activity, can activate TLR3.…”

Section: Treatment-specific Factors That Can Drive Immunogenicity In ...mentioning

confidence: 99%

“… 75 The authors speculate that the vector is less immunogenic, but further studies are needed to confirm the potential immunological advantage. 75 dsRNA, formed when the AAV ITRs have promoter activity, can activate TLR3. 76 Engineering the vector to weaken or eliminate ITR promoter function may decrease dsRNA formation and mitigate the immune response triggered by TLR3 activation.…”

Section: Treatment-specific Factors That Can Drive Immunogenicity In ...mentioning

confidence: 99%

Immune Responses and Immunosuppressive Strategies for Adeno-Associated Virus-Based Gene Therapy for Treatment of Central Nervous System Disorders: Current Knowledge and Approaches

Prasad¹,

Dimmock

Greenberg³

et al. 2022

Human Gene Therapy

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Adeno-associated viruses (AAVs) are being increasingly used as gene therapy vectors in clinical studies especially targeting central nervous system (CNS) disorders. Correspondingly, host immune responses to the AAV capsid or the transgene-encoded protein have been observed in various clinical and preclinical studies. Such immune responses may adversely impact patients' health, prevent viral transduction, prevent repeated dosing strategies, eliminate transduced cells, and pose a significant barrier to the potential effectiveness of AAV gene therapy. Consequently, multiple immunomodulatory strategies have been used in attempts to limit immune-mediated responses to the vector, enable readministration of AAV gene therapy, prevent end-organ toxicity, and increase the duration of transgene-encoded protein expression. Herein we review the innate and adaptive immune responses that may occur during CNS-targeted AAV gene therapy as well as host- and treatment-specific factors that could impact the immune response. We also summarize the available preclinical and clinical data on immune responses specifically to CNS-targeted AAV gene therapy and discuss potential strategies for incorporating prophylactic immunosuppression regimens to circumvent adverse immune responses.

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“…A recent report of a mouse study indicates that CpG motifs can be depleted from the ITRs without compromising the biological activity of the vector. 152 An alternative strategy, known as TLR9 cloaking, is to incorporate short DNA oligonucleotides (TLR9i) into the vector genome to antagonize TLR9 activation, potentially by outcompeting the CpG motifs. 153 Another option is to use cis-acting regulatory elements within the rAAV transgene to tailor tissue specificity and the level of transgene expression.…”

Section: Engineering the Raav Capsid And Genome To Reduce Immunogenicitymentioning

confidence: 99%