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AAV Capsid Assembly Using Escherichia coliDinh To Le, Marco T. Radukic, Kristian M. Müller*
Department of Cellular and Molecular Biotechnology, Bielefeld University, Bielefeld, Germany, * Corresponding author: [email protected]
[1] Le, D.T., Radukic, M.T. & Müller, K.M. Adeno-associated virus capsid protein expression in Escherichia coli and chemically definedcapsid assembly. Sci Rep 9, 18631 (2019). https://doi.org/10.1038/s41598-019-54928-y
The authors thank the Structural Biochemistry group (Prof. Niemann) and the ExperimentalBiophysics & Applied Nanoscience group (Prof. Anselmetti) at Bielefeld University for helpwith dynamic light scattering and atomic force microscopy measurements.
synTbio
IntroductionAAV vectors combine many properties that are useful in therapeutic
applications•AAVs lack pathogenicity and toxicity.
•AAVs show very low host immune response.
•AAV virus-like particles (VLPs) have been used in vaccinationexperiments.
•Recombinant AAV (rAAV) gene therapy products (Glybera,Luxturna and Zolgensma) are approved by EMA and FDA.
•AAP2 was produced in theform of inclusion bodies inE. coli (Fig. 6A) and added tothe in-vitro assemblyreaction.
• AAP2 improved AAV2 VLPformation in vitro by ELISA(Fig 6B).
•pET21a-AAP5 and pET24b-AAV5-VP3 were co-transformed into E. coli.AAP5 (33 kDa) and AAV5VP3 were successfully co-expressed in soluble form(Fig. 6C, 6D).
•The co-expression led to anincrease of AAV5 VLPs in E.coli (Fig. 6E).
Acknowledgement
References
Conclusion and Outlook•AAV2 VP3 protein can be expressed in E. coli and the protein can form AAV2VLPs in a chemically defined reaction[1].
•AAV5 VP3 protein can assemble VLPs within E. coli.
•AAP supports AAV capsid formation in an in-vitro reaction and inside E. coli.
•AAV VLPs can be used in various biomedical applications and may also serveas starting point for DNA loading.
Fig. 6 AAV2 VLPs - (A) SDS-PAGE analysis of AAP2 expression (lane 1) andpurification (lane 2). (B) AAP2 improved AAV2 capsid formation in vitro(ELISA with A20 antibody).
AAV5 VLPs - (C), (D) Western blot analysis of AAP5_His6 and AAV5VP3 co-expression in E. coli. (E) Number of purified VLPs in the VP3expression only and the co-expression system.
Fig. 1 (A) Radially color-cuedsurface representation of AAV2PDB ID #1LP3. (B) Genomicstructure of wild-type Adeno-associated virus serotype 2(AAV2).
AAV biology
•A member of the family Parvoviridae genusDependoparvovirus.
•AAV is a non-enveloped capsid of 60 proteins(VP1:VP2:VP3=1:1:10) (about 22 nm diameter) (Fig. 1A). VP3 asthe main capsid protein can form VP3 only capsids. AAPsupports capsid formation.
•The 4.7-kb ssDNA genome of AAV consists of two major openreading frames flanked by ITR sequences (Fig. 1B).
•AAV2 is the best studied serotype.
•AAV5, an AAP-independent serotype, is the most geneticallydivergent AAV serotype among known 13 human and non-human primate serotypes.
Capsid assembly and characterization of AAV VLPs
•AAV2 VLPs capsidassembly was carriedout by dialyzing VP3protein againstphosphate bufferedsaline (PBS) containing0.2 M L-arginine.
•AAV2 VLPs werecharacterized by ELISA(Fig. 4A), AFM (Fig. 4B)and dynamic lightscattering (Fig. 4C)[1].
•AAV5 VLPs assembledinside E. coli weredetected by ELISA (Fig.4D) and imaged byAFM (Fig. 4E, 4F).
Fig. 4 AAV2 VLPs - (A), (B), (C) ELISA (rAAV2 and AAV2 VLPs) probed withanti-AAV2 (intact capsid) antibody (A20 and A20scFv), AFM analysis ofAAV2 VLPs and dynamic light scattering analysis of AAV2 VLPs, respectively.
AAV5 VLPs - (D), (E), (F) ELISA probed with anti-AAV5 (intact capsid)antibody (ADK5a), AFM image of AAV5 VLPs and height profile of AAV5VLPs compared to rAAV5 produced in HEK293, respectively.
Rep 78 VP1
Rep68
Rep52
Rep40 AAP
VP2
VP3
rep cap (VP 123)
•HeLa cells were used forcellular uptake of AAV VLPsevaluation.
•Internalization of AAV VLPswas observed by fluorescentmicroscopy after 2 hours.
•Both in-vitro AAV2 VLPs[1] andAAV5 VLPs produced in E. colican internalize into HeLa cells(Fig. 5C).
Fig. 5 Internalization of rAAV5 and AAV5 VLPs. HeLa cells weretreated with (A) buffer, (B) rAAV5 or (C) AAV5 VLPs. Samples werestained with the ADK5a antibody followed by a Dylight 594conjugated secondary antibody (red) and DAPI (blue). Scale barsare 10 µm.
AAP improves capsid formation
AAV VLPs
•VLPs in general have been produced in E. coli and proposed for vaccination and drugdelivery.
•AAV VLPs were produced in HEK293 cells, Saccharomyces cerevisiae and from capsidproteins in the presence of cell extracts.
•rAAV production in HEK293 and Sf9 cells remains challenging. AAV VLPs could beused as a starting material for DNA loading in gene therapy.
A
B
Cellular uptake of AAV VLPs
Technische Fakultät
•AAV5 VP3 protein wasexpressed in the form ofinclusion bodies in E. coliBL21 (DE3) at 37oC andlargely soluble form at 18oC(confirmed by western blotanalysis using B1 antibody)(Fig. 3A).
•At 18oC expression, AAV5VLPs were detected inside E.coli BL21(DE3) harboringpET24b-AAV5-VP3 vector(Fig. 3B).
•AAV5 VLPs produced in E.coli can be purified by aPOROS AAVX resin column(Fig. 3C).
Expression of AAV5 VP3 protein and AAV5 VLP purification
Fig. 3 (A) Western blot analysis of AAV5 VP3 expression(whole-cell proteins (W), soluble fraction (S) and insolublefraction (I) of intracellular proteins). (B) Dot blot of E. coli cellextracts with anti-AAV5 (intact capsid) antibody (ADK5a) (E.coli and E. coli containing pET24b-AAV5-VP3 vector cellextracts). (C) SDS-PAGE analysis of AAV5 VLPs sample afterpurification by a POROS AAVX resin column.
A
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46
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37oC, 4 h 18oC, 18 h
W S I W S I undiluted
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•AAV2 VP3 protein wassuccessfully expressed in theform of inclusion bodies in E.coli BL21 (DE3) at 18oC using apET24b vector (Fig. 2A).
•Protein identity was confirmedby western blot analysis usingB1 antibody, the VP specificmonoclonal antibody (Fig. 2B).
•AAV2 VP3 protein was purifiedby anion exchangechromatography underdenaturing condition (Fig. 2C).
•Purified AAV2 VP3 was thenused for a chemically definedassembly reaction[1].
Expression and purification of AAV2 VP3 protein
Fig. 2 (A) SDS-PAGE analysis of expression of AAV2 VP3expression (pre-induction (Pre), whole-cell proteins (W),soluble fraction (S) and insoluble fraction (I) of intracellularproteins) (62 kDa). (B) Western blot analysis of VP3. (C) SDS-PAGE analysis of VP3 purification.
a b c
A B C
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E. coli
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