Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors

Ran, Gai; Chen, X; Xie, Yilin; Zheng, Qin; Xie, Jinyan; Yu, Chenghui; Pittman, Nikea; Qi, Sixian; Yu, Fa‐Xing; Agbandje‐McKenna, Mavis; Srivastava, Arun; Chen, Ling

doi:10.1016/j.omtm.2020.03.007

Cited by 23 publications

(18 citation statements)

References 38 publications

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“… 10,13,18,23 - 28 In addition to transgene design, synthetic promoter/enhancer elements could affect comparative immunogenicity via effects on FVIII expression level or FVIII expressing cell type, which is affected by both vector tropism (eg, AAV serotype) and promoter specificity for a given transduced cell type. 15,19,29 - 34 Finally, natural and recombinant AAV particles are immunogenic in humans and higher AAV capsid loads can induce cellular stress and creation of a proinflammatory microenvironment in the target organ that alters the immunogenicity risk of the transgene product. 11 , 30-32 , 35 …”

Section: Introductionmentioning

confidence: 99%

Pharmacokinetic analysis identifies a factor VIII immunogenicity threshold after AAV gene therapy in hemophilia A mice

et al. 2022

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Advances in the development of novel treatment options for hemophilia A are prevalent. However, the anti-FVIII neutralizing antibody (inhibitor) response to existing FVIII products remains a major treatment challenge. While some novel products are designed to function in the presence of inhibitors, they do not specific address the immunogenicity risk or mechanistic causes of inhibitor development, which remain unclear. Furthermore, most preclinical studies supporting clinical gene therapy programs have reported immunogenicity signals in animal models, especially at higher vector doses and sometimes using multiple vector designs. In these settings, immunogenicity risk factor determination, comparative immunogenicity of competing vector designs, and the potential for obtaining meaningful prognostic data remain relatively unexplored. Additionally, there remains the opportunity to investigate clinical gene therapy as an alternative to standard immune tolerance induction therapy. The current study was designed to address these issues through longitudinal dose-response evaluation of four AAV vector candidates encoding two different FVIII transgenes in a murine model of hemophilia A. Plasma FVIII activity and anti-FVIII antibody data were used to generate a pharmacokinetic model that 1) identifies initial FVIII expression kinetics as the dominant risk factor for inhibitor development, 2) predicts a therapeutic window where immune tolerance is achieved, and 3) demonstrates evidence of gene therapy-based immune tolerance induction. While there are known limitations to the predictive value of preclinical immunogenicity testing, these studies can uncover or support the development of design principles that can guide the development of safe and effective genetic medicines.

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Section: Introductionmentioning

confidence: 99%

Pharmacokinetic analysis identifies a factor VIII immunogenicity threshold after AAV gene therapy in hemophilia A mice

et al. 2022

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“…A recent study suggests that library-selected capsids such as LK03 can be further improved by additional site-directed mutagenesis. 47 Therefore, it is likely that our newly generated capsid with high tropism for hepatocytes can still be further improved. Earlier studies found that elimination of certain tyrosine residues on the surface of the AAV2 capsid could improve transduction and reduce major histocompatibility complex (MHC) class I presentation of capsid antigen.…”

Section: Discussionmentioning

confidence: 99%

Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity

Biswas

Marsic

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo , AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.

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“…This DNA shuffling approach has also successfully identified novel capsids with altered tropism, the ability to evade neutralizing antibodies, or both. For example, AAV-DJ, a widely used chimera of serotypes 2, 8, and 9, was selected for its ability to evade neutralizing antibodies relative to other serotypes [ 66 ], and AAV-LK03, a chimera of 7 different serotypes, was selected for its ability to transduce human hepatocytes in humanized FRG mice [ 70 ], and is currently being evaluated in a clinical trial for hemophilia A [ 71 ]. In summary, directed evolution of AAV offers researchers the capability to select capsids from large and highly diverse mutant capsid libraries that provide distinct advantages over existing vectors, and has led to the identification of many new AAV capsids with beneficial properties such as improved transduction efficiency, reduced immunogenicity, broadened tissue tropism, or refined tissue tropism for specific cell populations [ 62 , 65 , 66 , 72 – 77 ].…”

Section: Section I: Approaches To Identify Optimal Aav Capsidsmentioning

confidence: 99%

“…In an in vivo vector specificity analysis in humanized FRG mice, they showed that AAV-LK03 exhibits a stronger tropism for human hepatocytes in humanized mouse livers than AAV8, and AAV-LK03 is now being evaluated in a phase I/II clinical trial to treat hemophilia A (ClinicalTrials.gov: NCT03003533). Early results from this trial indicate that albeit safe at lower doses, high doses resulted in severe adverse events and subsequent loss of transgene expression in patients [ 71 ]. Recently, a rational design approach has been used to optimize AAV-LK03 for gene therapy applications via site-directed mutagenesis of surface-exposed residues.…”

Section: Section Ii: Identification Of Liver-tropic Aav Vectorsmentioning

confidence: 99%

Optimization of AAV vectors to target persistent viral reservoirs

Colón-Thillet

Jerome

Stone

2021

Virol J

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Gene delivery of antiviral therapeutics to anatomical sites where viruses accumulate and persist is a promising approach for the next generation of antiviral therapies. Recombinant adeno-associated viruses (AAV) are one of the leading vectors for gene therapy applications that deliver gene-editing enzymes, antibodies, and RNA interference molecules to eliminate viral reservoirs that fuel persistent infections. As long-lived viral DNA within specific cellular reservoirs is responsible for persistent hepatitis B virus, Herpes simplex virus, and human immunodeficiency virus infections, the discovery of AAV vectors with strong tropism for hepatocytes, sensory neurons and T cells, respectively, is of particular interest. Identification of natural isolates from various tissues in humans and non-human primates has generated an extensive catalog of AAV vectors with diverse tropisms and transduction efficiencies, which has been further expanded through molecular genetic approaches. The AAV capsid protein, which forms the virions' outer shell, is the primary determinant of tissue tropism, transduction efficiency, and immunogenicity. Thus, over the past few decades, extensive efforts to optimize AAV vectors for gene therapy applications have focused on capsid engineering with approaches such as directed evolution and rational design. These approaches are being used to identify variants with improved transduction efficiencies, alternate tropisms, reduced sequestration in non-target organs, and reduced immunogenicity, and have produced AAV capsids that are currently under evaluation in pre-clinical and clinical trials. This review will summarize the most recent strategies to identify AAV vectors with enhanced tropism and transduction in cell types that harbor viral reservoirs.

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Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors

Cited by 23 publications

References 38 publications

Pharmacokinetic analysis identifies a factor VIII immunogenicity threshold after AAV gene therapy in hemophilia A mice

Pharmacokinetic analysis identifies a factor VIII immunogenicity threshold after AAV gene therapy in hemophilia A mice

Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity

Optimization of AAV vectors to target persistent viral reservoirs

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