Neutralizing antibodies (NAbs) to adeno-associated virus (AAV) vectors are highly prevalent in humans 1,2 , block liver transduction 3-5 and vector readministration 6 , thus representing a major limitation to in vivo gene therapy. Strategies aimed at overcoming anti-AAV antibodies are being studied 7 , which often involve immunosuppression and are not efficient in removing pre-existing antibodies. Imlifidase (IdeS) is an endopeptidase able to degrade circulating IgG that is currently being tested in transplant patients 8 . Here we studied if IdeS can eliminate anti-AAV antibodies in the context of gene therapy. We showed efficient cleavage of pooled human IgG (IVIg) in vitro upon endopeptidase treatment. In mice passively immunized with IVIg, IdeS administration decreased anti-AAV antibodies and enabled efficient liver gene transfer.The approach was scaled up to non-human primates, a natural host for wild type AAV.IdeS treatment prior to AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector readministration. Finally, IdeS reduced anti-AAV antibody levels from human plasma samples in vitro, including plasma from prospective gene therapy trial participants. These results provide a potential solution to overcome pre-existing antibodies to AAV-based gene therapy.

Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.ADAMTS13 | von Willebrand factor | thrombotic thrombocytopenic purpura | hydrogen exchange | autoimmunity A cquired thrombotic thrombocytopenic purpura (TTP) is characterized by severe thrombocytopenia and microangiopathic hemolytic anemia, resulting from disseminated microvascular thrombosis. Patients with TTP may suffer from widespread organ damage, resulting in death if not aggressively treated (1, 2). In most patients, thrombotic microangiopathy is caused by IgG-type autoantibodies against the plasma metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) (3-5). ADAMTS13 regulates the function of the multidomain von Willebrand factor (VWF) (∼600 to ∼20,000 kDa) by cleaving it at the central A2 domain to regulate platelet-induced blood clot formation (3, 4).Immunological studies show that many acquired TTP patients with low plasma ADAMTS13 activity (less than 10%) harbor IgG autoantibodies that bind ADAMTS13 especially at the Cysrich and/or the spacer domain (4, 6). Deletion of these domains or grouped mutations therein can eliminate the binding of polyclonal anti-ADAMTS13 IgGs derived from many patients (4, 7-11). However, the exact ADAMTS13 binding sites of these autoantibodies are not known. Current treatment...

Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FVCD42b human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV HG/CD42b MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.

Brd2 is a member of the bromodomain-extraterminal domain (BET) family of proteins and functions as an acetyl-histone-directed transcriptional co-regulator and recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. While Brd2 acts as a protooncogene in mammalian blood, developmental studies link it to regulation of neuronal apoptosis and epilepsy, and complete knockout of the gene is invariably embryonic lethal. In Drosophila, the Brd2 homolog acts as a maternal effect factor necessary for segment formation and identity and proper expression of homeotic loci, including Ultrabithorax and engrailed. To test the various roles attributed to Brd2 in a single developmental system representing a non-mammalian vertebrate, we conducted a phenotypic characterization of Brd2a deficient zebrafish embryos produced by morpholino knockdown and corroborated by Crispr-Cas9 disruption and small molecule inhibitor treatments. brd2aMO morphants exhibit reduced hindbrain with an ill-defined midbrain-hindbrain boundary (MHB) region; irregular notochord, neural tube, and somites; and abnormalities in ventral trunk and ventral nerve cord interneuron positioning. Using whole mount TUNEL and confocal microscopy, we uncover a significant decrease, then a dramatic increase, of p53-independent cell death at the start and end of segmentation, respectively. In contrast, using qualitative and quantitative analyses of BrdU incorporation, phosphohistone H3-tagging, and flow cytometry, we detect little effect of Brd2a knockdown on overall proliferation levels in embryos. RNA in situ hybridization shows reduced or absent expression of homeobox gene eng2a and paired box gene pax2a, in the hindbrain domain of the MHB region, and an overabundance of pax2a-positive kidney progenitors, in knockdowns. Together, these results suggest an evolutionarily conserved role for Brd2 in the proper formation and/or patterning of segmented tissues, including the vertebrate CNS, where it acts as a bi-modal regulator of apoptosis, and is necessary, directly or indirectly, for proper expression of genes that pattern the MHB and/or regulate differentiation in the anterior hindbrain.