About

Valder R. Arruda, MD, PhD, is an Assistant Member at the Children's Hospital Research Institute at The Children's Hospital of Philadelphia, specializing in pediatrics. He holds an M.D. in Medicine with a focus on Hematology from the Faculty of Medicine of Triangulo Mineiro, Brazil, obtained in 1987, and a Ph.D. in Molecular Biology from the University of Campinas, Sao Paolo, Brazil, earned in 1995. His research interests center on developing gene-based strategies for treating bleeding and thrombotic diseases, with a particular focus on gene therapy for disorders of hemostasis and thrombosis. His laboratory has conducted early-phase clinical studies on adeno-associated viral (AAV) vectors for severe hemophilia B (factor IX deficiency) and is engaged in translational research to evaluate the efficacy and safety of intravascular delivery of AAV vectors to skeletal muscle or liver in animal models. Additionally, his work explores biological factors influencing AAV transduction and the risk of germline transmission. Arruda's research also investigates the role of the protein C anticoagulant pathway in coagulation and inflammation, aiming to identify novel therapeutic strategies for coagulation disorders and related diseases.

Research topics

• Medicine
• Internal medicine
• Chemistry
• Molecular biology
• Biology
• Biochemistry
• Chromatography

Selected publications

• Ancient origin of the furin sequence in the wolf F8 gene

  Open Veterinary Journal · 2025-01-01

  articleOpen access

  Background: Our previous characterization of canine coagulation factor VIII (FVIII) in the naturally occurring hemophilia A (HA) dog models provided insight into the evolution of the FVIII protein. We noted a unique sequence (H1645) in the R-X-X-R furin-recognition motif within the B domain of canine FVIII that was distinct from the sequence (R1645) in humans and other model organism species, including mice, rats, pigs, and sheep. R1645 was associated with lower secretion and biological activity, which can be improved with the canine H1645 sequence. Aim: Herein, we sought to determine the evolutionary origin of the canine H1645. Methods: ), in several free-range animals from diverse geographic locations and compared it to several breeds of domestic dogs. We also compared our sequences to publicly available reference sequences for other members of the class Mammalia, order Carnivora, and suborder Caniformia. Results: 19 X chromosomes from 12 gray wolves, at least 20 X chromosomes from 14 coyotes, and at least 12 X chromosomes from 12 domestic dogs of at least 10 distinct breeds all had the canine H1645 variant. Other members of the order Carnivora and suborder Caniformia, whose sequences are publicly available, had the R1645 sequence. Conclusion: Our results suggest that the H1645 variant in the furin-consensus sequence was likely derived after the infraorders Cynoidea and Arctoidea diverged but before the separation of the gray wolf and coyote and persists through the domestic dog.

  PublisherDOI

• Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

  UNC Libraries · 2024-07-23

  articleOpen access

  Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645-1648) in the design of B-domain-deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector-based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.

  PublisherDOI

• Analysis of vector genome integrations in multicentric lymphoma after AAV gene therapy in a severe hemophilia A dog

  Molecular Therapy — Methods & Clinical Development · 2023-11-14 · 7 citations

  articleOpen access

  Adeno-associated viral (AAV) vectors have traditionally been viewed as predominantly nonintegrating, with limited concerns for oncogenesis. However, accumulating preclinical data have shown that AAV vectors integrate more often than previously appreciated, with the potential for genotoxicity. To understand the consequences of AAV vector integration, vigilance for rare genotoxic events after vector administration is essential. Here, we investigate the development of multicentric lymphoma in a privately owned dog, PC9, with severe hemophilia A that was treated with an AAV8 vector encapsidating a B domain–deleted canine coagulation F8 gene. PC9 developed an aggressive B cell lineage multicentric lymphoma 3.5 years after AAV treatment. Postmortem analysis of the liver, spleen, and lymph nodes showed the expected biodistribution of the AAV genome. Integration events were found both in PC9 and a second privately owned hemophilia A dog treated similarly with canine F8 gene transfer, which died of a bleeding event without evidence of malignancy. However, we found no evidence of expanded clones harboring a single integration event, indicating that AAV genome integrations were unlikely to have contributed to PC9’s cancer. These findings suggest AAV integrations occur but are mostly not genotoxic and support the safety profile of AAV gene therapy. Adeno-associated viral (AAV) vectors have traditionally been viewed as predominantly nonintegrating, with limited concerns for oncogenesis. However, accumulating preclinical data have shown that AAV vectors integrate more often than previously appreciated, with the potential for genotoxicity. To understand the consequences of AAV vector integration, vigilance for rare genotoxic events after vector administration is essential. Here, we investigate the development of multicentric lymphoma in a privately owned dog, PC9, with severe hemophilia A that was treated with an AAV8 vector encapsidating a B domain–deleted canine coagulation F8 gene. PC9 developed an aggressive B cell lineage multicentric lymphoma 3.5 years after AAV treatment. Postmortem analysis of the liver, spleen, and lymph nodes showed the expected biodistribution of the AAV genome. Integration events were found both in PC9 and a second privately owned hemophilia A dog treated similarly with canine F8 gene transfer, which died of a bleeding event without evidence of malignancy. However, we found no evidence of expanded clones harboring a single integration event, indicating that AAV genome integrations were unlikely to have contributed to PC9’s cancer. These findings suggest AAV integrations occur but are mostly not genotoxic and support the safety profile of AAV gene therapy.

  PublisherOA PDFDOI

• HTRS2023.P2.13 Off-target toxicity of CART cell therapy in murine hemophilia A with inhibitors

  Research and Practice in Thrombosis and Haemostasis · 2023-08-01

  articleOpen access
  PublisherDOI

• Characteristics of BAY 2599023 in the Current Treatment Landscape ofHemophilia A Gene Therapy

  Current Gene Therapy · 2022-09-16 · 8 citations

  reviewOpen access

  Hemophilia A, a single gene disorder leading to deficient Factor VIII (FVIII), is a suitable candidate for gene therapy. The aspiration is for single administration of a genetic therapy that would allow the production of endogenous FVIII sufficient to restore hemostasis and other biological processes. This would potentially result in reliable protection from bleeding and its associated physical and emotional impacts. Gene therapy offers the possibility of a clinically relevant improvement in disease phenotype and transformational improvement in quality of life, including an opportunity to engage in physical activities more confidently. Gene therapy products for hemophilia A in advanced clinical development use adeno-associated viral (AAV) vectors and a codon-optimized B-domain deleted FVIII transgene. However, the different AAV-based gene therapies have distinct design features, such as choice of vector capsid, enhancer and promoter regions, FVIII transgene sequence and manufacturing processes. These, in turn, impact patient eligibility, safety and efficacy. Ideally, gene therapy technology for hemophilia A should offer bleed protection, durable FVIII expression, broad eligibility and limited response variability between patients, and long-term safety. However, several limitations and challenges must be overcome. Here, we introduce the characteristics of the BAY 2599023 (AAVhu37.hFVIIIco, DTX 201) gene therapy product, including the low prevalence in the general population of anti-AAV-hu37 antibodies, as well as other gene therapy AAV products and approaches. We will examine how these can potentially meet the challenges of gene therapy, with the ultimate aim of improving the lives of patients with hemophilia A.

  PublisherOA PDFDOI

• Immune complications and their management in inherited and acquired bleeding disorders

  Blood · 2022-07-06 · 20 citations

  articleOpen access1st authorCorresponding

  Disorders of coagulation, resulting in serious risks for bleeding, may be caused by autoantibody formation or by mutations in genes encoding coagulation factors. In the latter case, antidrug antibodies (ADAs) may form against the clotting factor protein drugs used in replacement therapy, as is well documented in the treatment of the X-linked disease hemophilia. Such neutralizing antibodies against factors VIII or IX substantially complicate treatment. Autoantibody formation against factor VIII leads to acquired hemophilia. Although rare, antibody formation may occur in the treatment of other clotting factor deficiencies (eg, against von Willebrand factor [VWF]). The main strategies that have emerged to address these immune responses include (1) clinical immune tolerance induction (ITI) protocols; (2) immune suppression therapies (ISTs); and (3) the development of drugs that can improve hemostasis while bypassing the antibodies against coagulation factors altogether (some of these nonfactor therapies/NFTs are antibody-based, but they are distinct from traditional immunotherapy as they do not target the immune system). Choice of immune or alternative therapy and criteria for selection of a specific regimen for inherited and autoimmune bleeding disorders are explained. ITI serves as an important proof of principle that antigen-specific immune tolerance can be achieved in humans through repeated antigen administration, even in the absence of immune suppression. Finally, novel immunotherapy approaches that are still in the preclinical phase, such as cellular (for instance, regulatory T cell [Treg]) immunotherapies, gene therapy, and oral antigen administration, are discussed.

  PublisherOA PDFDOI

• Leveraging CAR T cells to Achieve Desensitization and Enable Transplantation

  The Journal of Immunology · 2022-05-01

  article

  Abstract Pre-existing allo-antibodies (allo-Abs), that preclude transplant due to the risk of hyperacute rejection, lead to prolonged wait times and high mortality rates. Current desensitization approaches are ineffective as they do not adequately deplete allo-specific B cells and plasma cells (PCs). We hypothesize that stringent depletion of these cells is required to eliminate pre-existing allo-Abs. We leverage the exquisite ability of CAR T cells to eliminate target cells to desensitize transplant candidates. We constructed CARs targeting murine CD19 or BCMA, which cover the entire B cell-PC continuum. We first evaluated the function of CAR T cells against B cells and PCs in vitro. C57BL/6 mice were sensitized with BALB/c skin grafts. After skin rejection, sensitized mice received total body irradiation followed by treatment with either control T cells, CART-19 T cells, or a combination of CART-19 and CART-BCMA T cells (combo-CART). Allo-Abs, total Ig, and B cells were measured over 13 weeks. Functional desensitization was then assessed by induction of diabetes followed by BALB/c-derived islet cell transplant and glucose were measured to assess graft survival. CD19- and BCMA-targeted CARs effectively depleted primary B cells and PCs in vitro and in vivo. Control and CART-19 T cells were ineffective at desensitizing mice, but combo-CART treatment resulted in significant decrease of allo-Abs. Islet cell grafts succumbed to hyperacute rejection in 80% of control and CART-19 treated mice. However, combo-CART treatment resulted in prolonged graft survival in all mice (mean 35 days, range 16–60). Thus, CAR T cells targeting B cell and PC antigens represent a promising approach to desensitization and could enable lifesaving transplantation.

  PublisherDOI

• Long-Term Real-World Safety and Efficacy of Liver-Directed AAV Gene Therapy for Severe Hemophilia with and without Inbibitors in Privately Owned Dogs

  Blood · 2022-11-15

  article

  Background Hemophilia A (HA) and hemophilia B (HB) are inherited bleeding disorders resulting from deficiency of coagulation factors VIII (FVIII) or factor IX (FIX) activity, respectively. HA and HB both occur naturally in dogs. The bleeding phenotype of severe canine HA and HB is analogous to the human patient experience with frequent spontaneous and trauma-induced bleeding. Previous studies of dogs from HA and HB colonies at research institutes have been highly informative about the safety and efficacy of adeno-associated virus (AAV) liver-directed gene therapy for HA and HB. However, these animals are housed in highly controlled environments; in contrast, pet dogs with HA or HB are exposed to real-world challenges that likely affect their bleeding phenotype and the efficacy of novel therapeutics. In this regard, privately owned dogs may better recapitulate the human patient experience. Aims To determine the real-world efficacy of liver-directed AAV gene therapy for HA/HB in privately owned canines with and without inhibitors. Methods Eleven dogs with severe HA and one dog with severe HB were treated with liver-directed AAV gene therapy with canine (c) FVIII or cFIX, as detailed in Table 1. Pre-existing neutralizing antibodies to AAV8 were detected using a luciferase-based transduction inhibition assay. Pre- and post-gene therapy annualized bleeding rates were calculated by cumulative bleeding events divided by the age at gene therapy (pre) or duration of follow-up (post). FVIII activity was determined using a chromogenic assay and FIX activity using a one-stage assay. Results All dogs had AAV8 neutralizing antibody titers ≤ 1:5 at enrollment. The HA dogs (10 males and 1 female) were treated with either dual chain vectors of cFVIII-SQ (n = 2, total dose of 5 x 1013 vg/kg) or single-chain vectors encapsidating high-expression cFVIII variants: cFVIII-ΔF (n = 2, 6x1012 vg/kg) or cFVIII-ΔF/V3 (n = 7, 5.4-9 x 1012 vg/kg). F8 genetic testing revealed intron 22 inversions (n=7), small deletion (n=2), missense (n=1) and complex mutation (n=1) all compatible with severe HA. Of note, one HA dog (PC3) had a pre-existing FVIII inhibitor which was eradicated following gene therapy. In all dogs with available data (n=10), FVIII activity is > 1% with 5 in the moderate hemophilia range and 4 in the mild range with a median FVIII activity of 3.2% (range 1.1-13.8%) after a median duration of 4.1 years (range 2.6-8.9 years). In HA dogs, the ABR post-gene therapy declined by an average of 93%. The sole HB dog carried two missense F9 mutations and was treated with wildtype cFIX at 3 x 1012 vg/kg and has been followed for 4.5 years with FIX activity of 5.7% and has had no bleeding episodes since gene therapy. Three animals have died during follow up. The previously inhibitor positive dog (PC3) died approximately 2 years after gene therapy from an unrelated cause. An HA dog (PC1) treated with dual chain vector with expression in the ~2% normal range died from a bleeding episode at 8.8 years after gene therapy. Notably, many of his bleeds were associated with real-world activities including having his legs being pulled by a toddler. A third HA dog (PC9) was euthanized 3.5 years post-gene therapy after being diagnosed with widely disseminated cancer; the details of the vector integration analysis of his tumor will be reported elsewhere. Conclusion AAV gene therapy was successful at substantially ameliorating the bleeding phenotype in both male and females dogs with > 1 year of follow-up. However, some dogs continued to have bleeding, especially animals with <5% normal cFVIII activity. One animal developed cancer after gene therapy; there were no other safety concerns. As we have described previously, liver-directed AAV gene therapy also induced immune tolerance in an HA dog with a pre-existing inhibitors. We show that AAV liver-directed gene therapy is safe and efficacious in a real-world experience of privately owned dogs with severe HA and HB. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

  PublisherDOI

• Vector Analysis of Multicentric Lymphoma in a Severe Hemophilia Α Dog after AAV Gene Therapy

  Blood · 2022-11-15 · 3 citations

  article

  Background Adeno-associated viral (AAV) vector gene therapy for severe hemophilia A (HA) is approaching a clinical reality with multiple successful clinical trials. Despite this success, questions remain about the long-term safety of AAV vectors, especially regarding genomic integration and resulting oncogenesis. Studies of neonatal mice have demonstrated AAV integrations that drive hepatocellular carcinoma formation (Donsante et al. Science, 2007). Studies in HA canine models have also demonstrated AAV integrations and clonal expansion of hepatocytes with integrated vector genomes, but no evidence of tumor formation (Nguyen et al. Nat Biotechnol, 2021). Additionally, three tumors have been reported in AAV gene therapy recipients with HA or hemophilia B in clinical trials, without evidence of vector integrations driving oncogenesis. Though overall reassuring, these data highlight that the field needs to remain vigilant about the possibility of AAV integrations driving cancer development. Thus, the assessment of tumors in gene therapy recipients remains important for the AAV field. Aims To determine the role of AAV vector in the development of multicentric lymphoma in a severe HA dog that received liver-directed gene therapy. Methods Vector genome copy numbers in relevant tissues were determined by real-time PCR with primers situated in the 3'-end of the promoter and the 5'- end of the transgene. The vector copy number was calculated by comparing the analyte amplification signal against a standard curve of the plasmid DNA. Results A privately owned mixed-breed dog (PC9) with a history of recurrent spontaneous bleeding events presented with canine factor VIII (cFVIII) levels <1% normal, consistent with severe HA. At 9-months of age, he received liver-directed AAV8 gene therapy with a high-expressing canine factor FVIII (cFVIII-ΔF/V3) variant transgene at a dose of 6 x 1012 vg/kg via intravenous administration. Following gene therapy, his cFVIII levels increased to 2-3% normal and remained stable through duration of follow-up. His annualized bleeding rate decreased from 8 pre-gene therapy to 0 post-gene therapy. At 3.5 years after vector administration, PC9 was diagnosed with a widely disseminated cancer and euthanized due to complications of the disease. At autopsy, he was diagnosed with multicentric lymphoma involving peripheral and visceral lymph nodes, spleen, liver, and lungs. The average age of lymphoma diagnosis in dogs is between 6-9 years of age (Edwards et al. Vet Comp Oncol, 2003), so the development of cancer at an early age in a gene therapy recipient warranted additional study. Vector copy number analysis showed that the liver had the highest levels of AAV vector genomes, as expected. Liver samples showed a heterogenous distribution of vector genomes, consistent with previous studies of AAV8 transduction (Bell et al. Hum Gene Ther, 2011). Splenic samples also had a high level of AAV vector genomes, which again is consistent with known targeting of AAV8. Importantly, the lymph nodes had no detectable AAV vector genomes present, suggesting that vector integration could not contribute to lymphoma development. cFVIII levels remained stable before and after diagnosis of lymphoma, supporting lack of vector integration in the malignant cells. Conclusions AAV gene therapy resulted in a 100% reduction in the annualized bleeding rate in this pet dog with severe HA. Furthermore, the multicentric lymphoma that developed 3.5 years after liver-directed gene therapy is unlikely related to the AAV vector. We are unable to rule out other factors that may have contributed, including environmental influences or genetic predispositions for lymphoma. These results support the ongoing efforts to treat HA with AAV-based gene therapy and reinforce the safety profile of AAV vectors outside of a laboratory environment.

  PublisherDOI

• Ontogeny of the Alloimmune Anti-Canine Factor VIII Inhibitor Response in Severe Hemophilia Α Dogs

  Blood · 2021-11-05

  article

  Abstract The development of inhibitors to Factor VIII (FVIII) is a major complication of hemophilia A (HA) treatment. Most preclinical studies in HA animals have been limited to a xenoprotein response, e.g., evaluating the murine immune response to human FVIII (hFVIII). Severe HA dogs are a naturally occurring outbred model that recapitulates the spontaneous bleeding phenotype of the disease; their severe HA is due to intron 22 inversion (INV22) of the canine F8 gene, analogous to the INV22 found in 45% of severe HA patients. Importantly, similar to HA INV22 patients, about 20% of these animals develop an anti-canine FVIII (cFVIII) inhibitor response. This ability of the HA dogs to develop inhibitors in a species-specific manner provides an opportunity to probe the molecular ontogeny of an alloimmune anti-FVIII response. Here, we use antibody phage display to capture the humoral anti-cFVIII IgG repertoire in a dog with severe HA. This dog developed and maintained high titers of cFVIII inhibitor (89 Bethesda Units, BU) after exposure to cFVIII protein. The dog then received AAV liver-directed gene therapy encoding a cFVIII transgene, and after an initial anamnestic response characterized by rapid increase of cFVIII inhibitor titer (peak titer of 182 BU), the dog exhibited successful inhibitor eradication and immune tolerance induction, similar to our previous report (Finn et al., Blood 2010). Canine peripheral blood mononuclear cells were used to construct a single-chain variable fragment (scFv) phage display library which was sequentially selected four times against immobilized recombinant B-domain deleted (BDD) cFVIII protein. The amount of enrichment per round of selection plateaued during the third round from which 55 scFv/phage clones were isolated for characterization. Individual phage clones were sequenced by Sanger sequencing and screened for binding to both canine and human BDD-FVIII by ELISA. ScFv/phage clones were tested for inhibitory activity in a modified Bethesda assay. We identified 26 distinct scFv clones binding cFVIII based on heavy chain/light chain composition comprising 16 distinct CDRH3 sequences and 23 distinct CDRL3 sequences. The heavy chains of the 26 clones were derived from six canine germline IGHV genes, namely IGHV3-2, IGHV3-5, IGHV3-9, IGHV3-19, IGHV3-38, and IGHV3-41. The 16 distinct CDRH3 sequences had a mean length of 10.1 ± 3.6 amino acids, shorter than the 13.5 ± 3.6 amino acids previously reported for total canine CDRH3 repertoires (Steiniger et al., Mol. Immunol. 2014). Only 10 of the 26 FVIII-specific clones contained a lambda light chain, despite canine IgG repertoires dominated by the use of lambda light chains (Steiniger et al.). Eight of the 26 clones exhibited inhibitory activity. Although this dog was never exposed to hFVIII, 12/26 (46%) clones bound both cFVIII and hFVIII, consistent with the high degree of homology between the two orthologues with 85% identity. For three clones, framework region mutations and different light chain pairings resulted in altered inhibitory activity and hFVIII binding. Interestingly, one clone in particular comprised the majority of randomly screened scFv from the third and fourth rounds of phage library selection. These results suggest that cFVIII-specific B cells arise from multiple germline VH genes and exhibit high CDRH3 diversity. While the IgG-FVIII interaction has previously been suggested to be mostly influenced by the IgG heavy chain, our data suggest that for some antibodies, the IgG light chain may influence both inhibitory activity and epitope reactivity. Future studies will assess B cell clonal evolution via deep sequencing and longitudinal epitope specificity during multi-year immune tolerance induction by continuous exposure to cBDD-FVIII gene therapy. This is the first in-depth assessment of FVIII immunogenicity in a large HA model that avoids the use of a non-species specific antigen. Direct comparison with ongoing studies in HA inhibitor patients will inform the immunodominant epitopes of FVIII across species and thus provide insights on FVIII immunogenicity. Disclosures Doshi: Janssen: Consultancy; Spark Therapeutics: Speakers Bureau. Samelson-Jones: Spark: Research Funding; Pfizer: Consultancy, Research Funding. Siegel: Verismo Therapeutics, Inc: Other: Co-Founder and Equity Holder; Vetigenics, LLC: Other: Co-Founder and Equity Holder.

  PublisherDOI

Recent grants

• Hemostasis and Thrombosis: Chemistry, Biology and Physiology

  NIH · $33.0M · 2018–2029

• NIH Grant K01DK060580

  NIH · $290k · 2005

• NIH Grant R01HL084220

  NIH · $1.1M · 2009

• NIH Grant P01HL064190

  NIH · $25.4M · 2017

• Engineering cellular immunotherapy to modulate immune responses in hemophilia

  NIH · $1.7M · 2017–2023

Frequent coauthors

• Katherine A. High

  Rockefeller University

  292 shared

• Roland W. Herzog

  Indiana University – Purdue University Indianapolis

  161 shared

• Timothy C. Nichols

  106 shared

• Linda B. Couto

  Spark Therapeutics (United States)

  96 shared

• Rodney M. Camire

  University of Pennsylvania

  92 shared

• Paul Fields

  Adaptive Biotechnologies (United States)

  70 shared

• Joerg Schuettrumpf

  Biotest (Germany)

  65 shared

• Benjamin J. Samelson‐Jones

  Children's Hospital of Philadelphia

  65 shared

Labs

• Children's Hospital Research Institute, The Children's Hospital of PhiladelphiaPI