Patent Innovations

  • Capsid engineering of adeno-associated virus (AAV) vectors

    Posted on Mar 11, 2023

    Invention: The researchers from Adverum Biotechnologies. (Redwood City, CA) have developed a method for generating eukaryotic cell libraries for screening mutant AAV capsid proteins.

    Background of the invention

    Delivering therapeutic drugs via viral vectors is a potential strategy for treating and preventing various human diseases and genetic disorders. Since wild-type AAVs are nonpathogenic and have no etiologic relationship with any known disorders, recombinant vectors based on it are recognized to have great clinical safety. In human clinical trials, AAVs have demonstrated great promise. However, cell tropism (the capacity for viruses to enter and infect cells) and neutralizing antibody (NAb) reactions are the challenges for AAV-based gene therapy. Future studies will require better screening technologies as well as effective anti-immune therapy.

    Description of the invention

    Cell libraries designed to screen mutant AAV capsid proteins can be used for in vitro experiments and in vivo screening to find and select virions with particular and/or advantageous characteristics. The variant AAV virions may contain a variety of unique mutations in one or more of AAV capsid proteins (VP1, VP2, and/or VP3), which may give one or more of the virions desirable properties. Specifically, the goal of generating his library was to identify mutations that exhibit:

    1. increased or decreased heparan sulfate binding affinity relative to wild-type AAV
    2. increased infectivity of a AAV resistance cell
    3. increased evasion of neutralizing antibodies
    4. increased ability to cross an endothelial cell layer
    5. increased ability to cross the inner limiting membrane (ILM; the boundary between the retina and vitreous body).

    Methods for AAV capsid engineering

    The methods for creating stable cell lines and for screening mutant AAV capsid proteins are described below and summarized in Fig. 1.

    Generating a Packaging Cell Library

    A two-plasmid transfection strategy was used to create a library of packaging cells. The first plasmid contained a mutant capsid gene (mCap), which is linked to a eukaryotic promoter (CMV), flanked by inverted terminal repeats (ITRs) and a native attB attachment site, which is an integrase (φC31)-specific DNA attachment site in the genome. The second plasmid contains the φC31 integrase gene. A sequence encoding a green fluorescent protein (GFP) operably coupled to a different promoter was also present in the cassette to aid in the later identification and sorting of transfection and infected cells. The cell library was plated so that after 3 days, the cells are at 85–90% confluency, to produce virions for screening.

    Fig.1: Methods for generating mutant cell and AAV libraries.

    Generation of AAV for screening

    The cell library was co-transfected with an adenovirus-helper plasmid and adenovirus replication protein-2 (Rep-2)- expressing plasmid. After about five days, mutant AAVs were isolated and were used for screening.

    Controlling integration frequency for preventing cross packaging 

    Minimizing or reducing the cross packing of one mutant capsid gene with another mutant gene is a prerequisite for generating mutant AAV library. Cross packaging limits the ability to screen and select AAV mutants with desirable properties such as tropism, antibody reactivity etc., and may also result in the selection of false-positives and loss of the actual mutant/s. This can be prevented by developing a method for single integration of one capsid gene per cell. The integration frequency can be dependent on the ratio of mutant plasmids harboring the attB site to integrase-encoding plasmids. This was evaluated by transfecting HEK293 cells with two donor plasmids in conjunction with a third plasmid that encoded the integrase φC3. The first and second donor plasmid contains genes for red fluorescent protein (RFP) and green fluorescent protein (GFP), respectively. Additionally, both donor plasmids contain neomycin resistance gene for drug selection and attB DNA attachment site for genome integration. The results of these experiments have shown that a 1:50 ratio of donor plasmid to integrase φC3 plasmid has generated the highest amount of donor plasmid integration with less than 10% double-integration rate.

    In Vivo Screening of mutant AAV Virions for the Ability to Pass Through the Inner Limiting Membrane (ILM) and Infect Retinal Cells in Non-Human Primate Eyes 

    Naturally occurring serotypes of AAV are unable to cross through the ILM when AAV is administered via intravitreal injection in the eye. Therefore, naturally occurring AAVs cannot effectively transduce photoreceptor cells in the eye. To select mutant AAVs with improved photoreceptor targeting via intravitreal injection, a sample of mutant AAV capsid library (shown in Fig. 1) packaged mutant AAV virions, which contain random insertion of peptide in a surface exposed region of the capsid, were injected intravitreally into the eyes of African Green monkeys. After six weeks, retinal tissue explants from sacrificed animals were collected and transduced with the adenovirus serotype-5 (Ad5). The Ad5 increased the amount of mutant capsid RNA in infected cells by increasing the production of mutant AAV RNA, which encoded for mCap, in target cells that have been transduced with the mutant AAV. Then, RNA was extracted from tissue punches, the mutant capsid DNA was amplified using RT-PCR, and the samples were sequenced to identify the mutant AAV capsids that could successfully traverse the ILM and infect retinal cells. The mutant library can be repackaged in AAVs and continually screened for mutant AAVs until a desired mutant capsid protein has been identified. The details of this screening method are shown in Fig. 1

    Medical applications of the invention 

    The methods described in this patent application can be used for developing novel AAV vectors for gene therapy. 


    Chavez, et. al., Mutant viral capsid libraries, and related systems and methods. United States Patent Application 11,427,931; August 30, 2022


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