AAV Entry: Filling in the Blanks
Authors: Hildegard Büning
Journal: Molecular Therapy (2020)

In this issue of Molecular Therapy, Dudek et al.1Dudek A.M. Zabaleta N. Zinn E. Pillay S. Zengel J. Porter C. Franceschini J.S. Estelien R. Carette J.E. Zhou G.L. Vandenberghe L.H. GPR108 Is a Highly Conserved AAV Entry Factor.Mol. Ther. 2020; 28: 367-381Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar report the discovery that GPR108 is an essential entry factor for adeno-associated virus (AAV) vectors. In the absence of GPR108, transduction of human and murine cell lines as well as primary cells was severely impaired. This also held true in vivo, where, depending on the serotype used, 10-fold to 100-fold reduced transgene expression was observed in GPR108 knockout mice compared to wild-type controls following retro-orbital administration. This study by Dudek et al.1Dudek A.M. Zabaleta N. Zinn E. Pillay S. Zengel J. Porter C. Franceschini J.S. Estelien R. Carette J.E. Zhou G.L. Vandenberghe L.H. GPR108 Is a Highly Conserved AAV Entry Factor.Mol. Ther. 2020; 28: 367-381Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar is a seminal contribution that begins to fill in the blanks of our knowledge of how AAV reaches the nucleus to deliver its genetic cargo. When the first AAV serotype was discovered as a contamination in adenoviral preparations more than 50 years ago,2Atchison R.W. Casto B.C. Hammon W.M. Adenovirus-Associated Defective Virus Particles.Science. 1965; 149: 754-756Crossref PubMed Scopus (559) Google Scholar the success of vectors derived thereof in gene and cell therapy, genome editing, and basic research could not even remotely be envisioned. Since 2012, when Glybera obtained marketing approval for the treatment of lipoprotein lipase deficiency as the first gene therapy in the Western world, gene and cell therapy has experienced tremendous academic and commercial interest. In the meantime, two additional gene therapy drugs relying on AAV vectors as the delivery tool entered the market (Luxturna for the treatment of Leber congential amaurosis and Zolgensma for the treatment of spinal muscular atrophy),3Keeler A.M. Flotte T.R. Recombinant Adeno-Associated Virus Gene Therapy in Light of Luxturna (and Zolgensma and Glybera): Where Are We, and How Did We Get Here?.Annu. Rev. Virol. 2019; 6: 601-621Crossref PubMed Scopus (136) Google Scholar and about 170 interventional clinical trials using this vector system are currently registered (https://clinicaltrials.gov/). The results reported so far from some of these trials are impressive, suggesting that more drug approvals are to be expected in the near future.3Keeler A.M. Flotte T.R. Recombinant Adeno-Associated Virus Gene Therapy in Light of Luxturna (and Zolgensma and Glybera): Where Are We, and How Did We Get Here?.Annu. Rev. Virol. 2019; 6: 601-621Crossref PubMed Scopus (136) Google Scholar This success has been fostered by continuous efforts to optimize the AAV vector system through engineering of the vector genome and the capsid so as to improve vector manufacturing and enhance vector efficacy by deciphering details and players in the process of cell transduction. In particular, with regard to the latter, it is astonishing how many blank spots remain on our map of the early events in cell transduction, despite the prominent role of AAV in gene and cell therapy. Recent advances have become possible due to the emergence of novel screening and bioinformatic analysis technologies. In their study, Dudek et al.1Dudek A.M. Zabaleta N. Zinn E. Pillay S. Zengel J. Porter C. Franceschini J.S. Estelien R. Carette J.E. Zhou G.L. Vandenberghe L.H. GPR108 Is a Highly Conserved AAV Entry Factor.Mol. Ther. 2020; 28: 367-381Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar used a genome-wide CRISPR screen targeting protein-encoding genes and microRNAs (miRNAs) of the human hepatoma cell line, Huh7, and identified GPR108 as a second essential entry factor for AAV. This finding follows the re-discovery of AAVR (AAV receptor) in 2016.4Pillay S. Meyer N.L. Puschnik A.S. Davulcu O. Diep J. Ishikawa Y. Jae L.T. Wosen J.E. Nagamine C.M. Chapman M.S. Carette J.E. An essential receptor for adeno-associated virus infection.Nature. 2016; 530: 108-112Crossref PubMed Scopus (254) Google Scholar,5Mizukami H. Young N.S. Brown K.E. Adeno-associated virus type 2 binds to a 150-kilodalton cell membrane glycoprotein.Virology. 1996; 217: 124-130Crossref PubMed Scopus (81) Google Scholar In light of the extensive current activities reported from different groups (for example, at the 2019 annual meeting of the American Society for Gene and Cell Therapy), it is likely that further holes in our understanding of the AAV-host interaction will be filled soon. Since 2016, AAVR and its interaction with AAV has been studied intensively, arguing for a function in the trafficking of AAV following engagement at the cell membrane toward the trans-Golgi network (TGN). Importantly, Dudek et al.1Dudek A.M. Zabaleta N. Zinn E. Pillay S. Zengel J. Porter C. Franceschini J.S. Estelien R. Carette J.E. Zhou G.L. Vandenberghe L.H. GPR108 Is a Highly Conserved AAV Entry Factor.Mol. Ther. 2020; 28: 367-381Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar bolster this concept in that the results point toward AAVR and GPR108 serving as factors within the same pathway, with AAVR acting upstream of GPR108. Many, but not all, AAV serotypes and variants are dependent on AAVR and, interestingly, different serotypes bind to different domains of this protein.6Herrmann A.K. Grimm D. High-Throughput Dissection of AAV-Host Interactions: The Fast and the Curious.J. Mol. Biol. 2018; 430: 2626-2640Crossref PubMed Scopus (19) Google Scholar To increase the likelihood of identifying an entry factor distinct from AAVR, Dudek et al.1Dudek A.M. Zabaleta N. Zinn E. Pillay S. Zengel J. Porter C. Franceschini J.S. Estelien R. Carette J.E. Zhou G.L. Vandenberghe L.H. GPR108 Is a Highly Conserved AAV Entry Factor.Mol. Ther. 2020; 28: 367-381Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar selected rh32.33, a hybrid variant derived from two related rhesus macaque isolates,7Vandenberghe L.H. Breous E. Nam H.J. Gao G. Xiao R. Sandhu A. Johnston J. Debyser Z. Agbandje-McKenna M. Wilson J.M. Naturally occurring singleton residues in AAV capsid impact vector performance and illustrate structural constraints.Gene Ther. 2009; 16: 1416-1428Crossref PubMed Scopus (51) Google Scholar which represents an example of an AAV variant that transduces cells independently of AAVR. In addition, the hepatoma cell line Huh7 had been engineered to lack AAVR. Nevertheless, when re-analyzing data from the initial AAVR screen, GPR108 was already present among the hits.4Pillay S. Meyer N.L. Puschnik A.S. Davulcu O. Diep J. Ishikawa Y. Jae L.T. Wosen J.E. Nagamine C.M. Chapman M.S. Carette J.E. An essential receptor for adeno-associated virus infection.Nature. 2016; 530: 108-112Crossref PubMed Scopus (254) Google Scholar Even though the screening approach was restricted to rh32.33, it soon became obvious that GPR108 is widely used, i.e., that its function in AAV-mediated cell transduction is widespread. Specifically, in addition to rh32.33, other naturally-occurring serotypes across all AAV clades, a primary receptor binding-deficient variant (AAV2HSPG−), and tropism-modified AAV vectors such as AAV9.PHP.B or AAV2.7m8, as well as Anc80 (a synthetic AAV), all demonstrated impressive dependency on the presence of GPR108. As is the case for AAVR however, whereas most AAV variants seem to be dependent on GPR108, there are also exceptions, as exemplified by AAV5. AAV5 is highly divergent compared to other AAV serotypes,8Gao G. Vandenberghe L.H. Alvira M.R. Lu Y. Calcedo R. Zhou X. Wilson J.M. Clades of Adeno-associated viruses are widely disseminated in human tissues.J. Virol. 2004; 78: 6381-6388Crossref PubMed Scopus (766) Google Scholar is dependent on AAVR (but not GPR108) for cell transduction, and emerged—as did AAV4–before the divergence of Dependoparvoviridae into the two monophylic groups with distinct clades, i.e., AAV serotypes that share functional and serologic similarities previously defined by Gao et al.8Gao G. Vandenberghe L.H. Alvira M.R. Lu Y. Calcedo R. Zhou X. Wilson J.M. Clades of Adeno-associated viruses are widely disseminated in human tissues.J. Virol. 2004; 78: 6381-6388Crossref PubMed Scopus (766) Google Scholar and Lukashov et al.9Lukashov V.V. Goudsmit J. Evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses.J. Virol. 2001; 75: 2729-2740Crossref PubMed Scopus (113) Google Scholar Interestingly, some serotypes retain the ability to transduce certain cells in the absence of GPR108, albeit at a reduced level (e.g., AAV4 in primary mouse embryonic fibroblasts). The cellular function of GPR108 is unclear. Based on its sequence, GPR108 is a member of the family of G-protein coupled receptors, characterized by a hydrophobic signal peptide at the N terminus and seven transmembrane domains.10Dong D. Zhou H. Na S.Y. Niedra R. Peng Y. Wang H. Seed B. Zhou G.L. GPR108, an NF-κB activator suppressed by TIRAP, negatively regulates TLR-triggered immune responses.PLoS ONE. 2018; 13: e0205303Crossref PubMed Scopus (11) Google Scholar Interestingly, both N- and C-terminal domains, which are located on opposite sides of the membrane, are required to support AAV transduction, arguing that a signal initiated by binding of AAV to GPR108 might be transmitted from the lumen toward the cytoplasm. Alternatively, it could be envisioned that GPR108 supports a step in the transduction process that "bridges" the membrane (see below). The closest paralog of GPR108 is GPR107, used here as a control unable to complement for the loss of GPR108. GPR107 is responsible for the recycling of receptors from the endocytic compartment to the plasma membrane, thus being involved in the regulation of receptor-mediated endocytosis.11Zhou G.L. Na S.Y. Niedra R. Seed B. Deficits in receptor-mediated endocytosis and recycling in cells from mice with Gpr107 locus disruption.J. Cell Sci. 2014; 127: 3916-3927Crossref PubMed Scopus (15) Google Scholar GPR108, however, does not seem to have an impact on AAV uptake, since AAV vectors demonstrated comparable binding to membranes of GPR108 wild-type and knockout cells and were detectable in membrane, cytosol, and nuclear fractions. Dong et al.10Dong D. Zhou H. Na S.Y. Niedra R. Peng Y. Wang H. Seed B. Zhou G.L. GPR108, an NF-κB activator suppressed by TIRAP, negatively regulates TLR-triggered immune responses.PLoS ONE. 2018; 13: e0205303Crossref PubMed Scopus (11) Google Scholar characterized GPR108 as a negative regulator of Toll-like-receptor (TLR)-triggered immune responses. GPR108 co-localized with the Golgi markers Giantin and GM130 as well as with TLR3, -7, -4, and -9, an observation that was subsequently validated for TLR3, -4, and -7 by co-immunoprecipitation (TLR9 was not included).10Dong D. Zhou H. Na S.Y. Niedra R. Peng Y. Wang H. Seed B. Zhou G.L. GPR108, an NF-κB activator suppressed by TIRAP, negatively regulates TLR-triggered immune responses.PLoS ONE. 2018; 13: e0205303Crossref PubMed Scopus (11) Google Scholar The GPR108-TLR9 interaction might be of interest as this TLR is a well-described pattern recognition receptor for AAV vectors, which triggers a type I interferon response following recognition of AAV vector genomes, thereby initiating adaptive immune responses against the vector and/or the transgene product.12Zhu J. Huang X. Yang Y. The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice.J. Clin. Invest. 2009; 119: 2388-2398Crossref PubMed Scopus (208) Google Scholar Dudek et al.1Dudek A.M. Zabaleta N. Zinn E. Pillay S. Zengel J. Porter C. Franceschini J.S. Estelien R. Carette J.E. Zhou G.L. Vandenberghe L.H. GPR108 Is a Highly Conserved AAV Entry Factor.Mol. Ther. 2020; 28: 367-381Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar did not follow this line but confirmed TGN localization of GPR108 using TGN46 as a marker. Based on the GPR108-TGN46 colocalization, the authors postulated the presence of GPR108 in late endosomes and TGN. Through gain- and loss-of-function chimeras between AAV2 and AAV5, the authors mapped the unique region of AAV2 VP1 capsid protein (VP1u) that imparts GPR108 dependency. In acidifying endosomes, this domain is externalized from the particle and therefore only then accessible to cellular co-factors. The authors hypothesize that GPR108 plays a role in this VP1u-mediated endosomal (or TNG) escape. One option is that GPR108 stabilizes the extruded VP1u. VP1u harbors an intrinsic phospholipase activity that could form a lipolytic pore through which AAV particles could escape from the endosome.13Girod A. Wobus C.E. Zádori Z. Ried M. Leike K. Tijssen P. Kleinschmidt J.A. Hallek M. The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity.J. Gen. Virol. 2002; 83: 973-978Crossref PubMed Scopus (253) Google Scholar,14Stahnke S. Lux K. Uhrig S. Kreppel F. Hösel M. Coutelle O. Ogris M. Hallek M. Büning H. Intrinsic phospholipase A2 activity of adeno-associated virus is involved in endosomal escape of incoming particles.Virology. 2011; 409: 77-83Crossref PubMed Scopus (58) Google Scholar A direct role of GPR108 in pore formation was also posited, involving the release of vectors from the TGN rather than from the endosomal compartment. According to this scenario, AAVR, which acts upstream of GPR108, binds AAV particles at the cell membrane, and the AAVR-AAV complex traffics through the endosomal compartment toward the TGN. In the endosome, the extruded VP1u would then bind to GPR108, equipping AAV particles with the required support to deliver their genetic payload to the nucleus. If direct or indirect support of pore formation indeed represents the function of GPR108, then AAV5 must have evolved an alternate strategy to convey efficient endosomal or TGN escape because VP1u of AAV5 possesses phospholipase activity but does not bind GPR108. Further studies are required to understand more fully the function of GPR108 in the process of AAV transduction and to corroborate the proposed updated model of cell transduction. However, the results strongly argue that GPR108 acts at a step prior to nuclear entry and that it plays an essential function in promoting processes involved in transporting AAV to the nucleus, because significantly fewer vector genomes are detected in the nuclear compartment of GPR108 knockout compared to wild-type cells. Since the most divergent serotypes do not rely on GPR108 (AAV5) or are—under conditions yet to be defined— less dependent (AAV4), the GPR108-dependent promotion of nuclear delivery must have emerged at a time when the two monophylic groups were evolving. Thus, relations between members of Dependoparvoviridae might additionally be classified according to their dependency upon AAVR and/or GPR108. In addition, it would be interesting to identify the factor(s) that replace the function of AAVR in transduction by rh32.33 and AAV4, which both rely on GPR108, but not AAVR, while the converse is true for AAV5. The discovery of Dudek et al.1Dudek A.M. Zabaleta N. Zinn E. Pillay S. Zengel J. Porter C. Franceschini J.S. Estelien R. Carette J.E. Zhou G.L. Vandenberghe L.H. GPR108 Is a Highly Conserved AAV Entry Factor.Mol. Ther. 2020; 28: 367-381Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar is not only a seminal contribution to closing the gaps in our knowledge of the processes of AAV transduction, but it might also explain why certain cell types are refractory to AAV transduction. In addition, these findings will likely impact the development of the next generation AAV vectors, which now can be tailored to render them independent or dependent from this newly described AAV entry factor GPR108. GPR108 Is a Highly Conserved AAV Entry FactorDudek et al.Molecular TherapyNovember 12, 2019In BriefAdeno-associated virus (AAV) has been repurposed as a highly effective gene therapy vector, yet conserved cellular factors required for cellular entry are poorly understood. In this issue of Molecular Therapy, Vandenberghe and colleagues describe identification of a novel conserved AAV entry factor that is required in vivo for AAV transduction. Full-Text PDF Open Archive