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TRIM21 Mediates Antibody Inhibition of Adenovirus-Based Gene Delivery

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TRIM21 Mediates Antibody Inhibition of Adenovirus-Based Gene Delivery TRIM21 mediates antibody inhibition of adenovirus- based gene delivery and vaccination Maria Bottermanna, Stian Fossb,c,d, Laurens M. van Tienena, Marina Vaysburda, James Cruickshanka, Kevin O’Connella, Jessica Clarka, Keith Mayesa, Katie Higginsona, Jack C. Hirste, Martin B. McAdamc, Greg Slodkowicza, Edward Hutchinsone, Patrycja Kozika, Jan Terje Andersenb,c,d, and Leo C. Jamesa,1 aProtein and Nucleic Acid Chemistry Division, Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom; bDepartment of Biosciences, Centre for Immune Regulation, University of Oslo, N-0316 Oslo, Norway; cDepartment of Immunology, Centre for Immune Regulation, Oslo University Hospital, N-0372 Oslo, Norway; dDepartment of Pharmacology, Institute of Clinical Medicine, University of Oslo, N-0372 Oslo, Norway; and eMRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, United Kingdom Edited by Michael B. A. Oldstone, The Scripps Research Institute, La Jolla, CA, and approved August 14, 2018 (received for review April 13, 2018) Adenovirus has enormous potential as a gene-therapy vector, but after endocytosis and can therefore carry antibody into the cy- preexisting immunity limits its widespread application. What is tosol. Upon binding to the Fc region of IgG with its PRYSPRY responsible for this immune block is unclear because antibodies motif, TRIM21 becomes activated and sequentially recruits the potently inhibit transgene expression without impeding gene E2 enzymes Ube2W and Ube2N/Ube2V2, which N-terminally transfer into target cells. Here we show that antibody prevention polyubiquitinate TRIM21 with anchored K63 chains. This results of adenoviral gene delivery in vivo is mediated by the cytosolic in recruitment of the proteasome and rapid degradation of the antibody receptor TRIM21. Genetic KO of TRIM21 or a single- viral particle while, simultaneously, the proteasome-associated antibody point mutation is sufficient to restore transgene expres- deubiquitinase PohI liberates the K63-linked ubiquitin chains, – sion to near-naïve immune levels. TRIM21 is also responsible for which stimulate innate immune signaling pathways (15 18). Fur- blocking cytotoxic T cell induction by vaccine vectors, preventing thermore, degradation of the viral capsid results in the exposure of a protective response against subsequent influenza infection and the Ad5 DNA to the cytosolic DNA sensor cGAS, which can an engrafted tumor. Furthermore, adenoviral preexisting immunity initiate a second wave of innate immune signaling (19). Here we investigate what role TRIM21 has in the preexisting can lead to an augmented immune response upon i.v. administra- MICROBIOLOGY tion of the vector. Transcriptomic analysis of vector-transduced tis- immune block to Ad5 gene delivery in vivo and in the activation sue reveals that TRIM21 is responsible for the specific up-regulation of innate immune signaling. of hundreds of immune genes, the majority of which are compo- Results nents of the intrinsic or innate response. Together, these data As hexon is the immunodominant antigen in preexisting Ad5 define a major mechanism underlying the preimmune block to immunity, we chose as a model the anti-hexon mouse–human adenovirus gene therapy and demonstrate that TRIM21 efficiently chimeric IgG1 monoclonal antibody (mAb) 9C12 (rh9C12), blocks gene delivery in vivo while simultaneously inducing a rapid which binds two of the hexon HVRs (20). First, we investigated program of immune transcription. TRIM21 | adenovirus | host–pathogen | gene therapy | viral vector Significance Viral-based delivery vectors have huge potential in the treat- denoviral vectors are the most frequently used delivery ve- ment of human disease. Adenoviral vectors specifically have hicles in gene therapy clinical trials (1) and have enormous A proven highly efficacious in delivering corrected genes, as part potential as vaccine vectors and oncolytic agents in cancer of gene therapy, and vaccine epitopes for treating cancer and treatment (2). They are attractive because they can be grown to infectious disease. A principal obstacle to their widespread use high titers, their genome sequence can easily be manipulated, is that antibodies potently neutralize them, limiting treatment and they induce robust transgene expression in a variety of di- to naïve patients. How antibodies block adenovirus-based viding and nondividing cell types. Despite these advantageous transduction has long remained a mystery because, even though properties, the high level of preexisting immunity in the human they prevent transgene expression, they do not prevent transgene population prevents their widespread use. This is particularly delivery into target tissue. Here we show that the cytosolic anti- – true for adenovirus 5 (Ad5) (3 5), the subtype on which most body receptor TRIM21 is responsible for intercepting adenoviral adenoviral vectors have been based. Preexisting humoral immu- gene therapy and vaccine vectors and neutralizing them. Gene KO nity is a correlate of poor transgene expression (6) and, during of TRIM21 or a single-antibody mutation that prevents interaction vaccination, a subsequently diminished CD8 T cell response is sufficient to restore transgene expression. against the desired antigen (6–9). Therefore, knowing what me- diates the block to transgene expression and activates the immune Author contributions: M.B., S.F., L.M.v.T., J.C.H., E.H., P.K., J.T.A., and L.C.J. designed re- response in the presence of anti-Ad5 antibodies might provide search; M.B., S.F., L.M.v.T., M.V., J. Cruickshank, K.O., J. Clark, K.M., K.H., J.C.H., M.B.M., G.S., E.H., P.K., and J.T.A. performed research; M.B., S.F., L.M.v.T., M.V., and L.C.J. ana- insight into new approaches to circumvent preexisting immunity. lyzed data; and M.B. and L.C.J. wrote the paper. The majority of anti-Ad5 neutralizing Abs (NAbs) are directed The authors declare no conflict of interest. against the major coat protein hexon (5, 10, 11), and more spe- This article is a PNAS Direct Submission. cifically its solvent-exposed hypervariable regions (HVRs) (12). As This open access article is distributed under Creative Commons Attribution-NonCommercial- hexon is not involved in the coxsackie and adenovirus receptor NoDerivatives License 4.0 (CC BY-NC-ND). (CAR)-dependent entry mechanism that Ad5 uses, which is me- Data deposition: Raw and processed RNA sequencing data generated from this study diated by fiber and penton base (13, 14), the neutralizing effect is have been deposited in NCBI’s Gene Expression Omnibus and are accessible through unlikely to involve classical entry blocking. GEO Series accession no. GSE119119. We have previously shown that the cytosolic antibody receptor 1To whom correspondence should be addressed. Email: [email protected]. TRIM21 mediates the efficient cytosolic neutralization of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. antibody-bound Ad5 in vitro. Ad5 gains access to the cytosol 1073/pnas.1806314115/-/DCSupplemental. during infection through penetration of early endosomes shortly www.pnas.org/cgi/doi/10.1073/pnas.1806314115 PNAS Latest Articles | 1of6 Downloaded by guest on September 28, 2021 whether vector uptake in vivo was inhibited by rh9C12 via a liver-resident Kupffer cells by engaging FcγRs (21), which might receptor-blocking effect. Mice were injected with rh9C12 2–4h result in decreased infection. We produced a “LALA” (L234A/ before injection with Ad5 to mimic circulating humoral immu- L235A) mutant of rh9C12, which significantly diminishes FcγR nity to the virus. Livers were harvested 2–8 h postinfection, and binding by ablating the binding site (Fig. 1B) (22). The LALA quantitative PCR (qPCR) was used to quantify viral genomes mutation had no measurable impact on the block to transgene within the tissue. At no time point did we observe a difference in expression by rh9C12 (Fig. 1C), indicating that antibody in- hibition is not the result of rerouting virus to a noninfectious and genome delivery in the presence of antibody (Fig. 1A). To assess γ whether rh9C12 diminishes transgene expression, we looked at Fc R-dependent cell entry pathway. As we have previously demonstrated that TRIM21 mediates liver transduction by Ad5 carrying the luciferase gene (Ad5-Luc) potent postentry neutralization of Ad5 in cells (20, 23, 24), we and found that, although rh9C12 did not affect viral gene sought to determine whether TRIM21 is responsible. We pro- transfer, it diminished transgene expression 1,000-fold (Fig. 1C). duced a further variant of rh9C12 with mutation H433A, which A possible explanation for this observed discrepancy is that the ablates the interaction with TRIM21 (24). Remarkably, H433A presence of antibody alters transgene expression by changing the almost completely abolished the antibody block to Ad5 trans- entry route of virus into cells and preventing access to the cy- gene expression in the liver (Fig. 1D). Although the TRIM21 tosol. Antibody has previously been shown to allow uptake by binding site is distinct from that of FcγRs, it does overlap with that of the neonatal Fc receptor (FcRn) (Fig. 1B). To ensure that the phenotype we observed is not caused by a shorter serum t1/2 as a result of poorer FcRn-mediated recycling, we determined A BC1 that mouse FcRn binds WT rh9C12 and H433A with similar Ad5 efficiency (SI Appendix, Fig. S1 A and B). Furthermore, no dif- 105 Ad5 + rh9C12 0.1 ference in the serum levels of these antibodies was detected 24 h 104 post injection (SI Appendix,Fig.S1C), showing that there has been FcgR ns 103 0.01 no alteration in FcRn recycling, in accordance with previous reports binding site (25). We sought to confirm this result genetically by repeating the 2 TRIM21 10 Relative infection & FcRn 0.001 experiment with WT rh9C12 in Trim21 KO mice. Although overall 101 binding transduction levels in both strains were comparable (SI Appendix, site 100 0.0001 Fig. S2A), the T21-KO mice given rh9C12 closely phenocopied WT Viral copy number per 200ng DNA Viral 2h 4h 6h 8h mice given the H433A mutant in liver and spleen (Fig.
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