Browsing by Subject "Hemophilia"
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Item Adeno-associated virus-based gene therapy for hemophilia-addressing the gaps(Elsevier, 2024-12-31) Miesbach, Wolfgang; Batty, Paul; Chowdary, Pratima; Fong, Sylvia; Kaczmarek, Radoslaw; Leebeek, Frank W. G.; Long, Brian; Mahlangu, Johnny; Makris, Mike; Pierce, Glenn F.; Pipe, Steven W.; Srivastava, Alok; Voorberg, Jan; Peyvandi, Flora; Pediatrics, School of MedicineAdeno-associated virus-based gene therapy for hemophilia has emerged as a revolutionary treatment option, offering potential correction of clotting factor deficiency through a single intravenous infusion of functional genes directed to hepatocytes. With 3 gene therapies recently approved, this approach shows promise in transforming the lives of individuals with hemophilia. However, the complexity of gene therapy and the lack of standardization of methods in different components of this therapy can lead to unique challenges for clinical implementation. This manuscript follows literature reviews and structured discussions by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Working Group on Gene Therapy that identified specific areas requiring standardization of methods, including viral vector production, liver function assessment, quantification of factor (F)VIII and FIX expression levels, assessment of antiadeno-associated viral antibodies, and genomic integration detection methods. Standardization strategies aim to achieve consistent vector quality, effective patient selection, and uniform assessment methods by implementing advanced laboratory techniques and standardized protocols. Standardizing these parameters is essential for improving the understanding of short-term and long-term safety and efficacy of gene therapy in hemophilia. This effort aims to enhance the predictability of individual responses, address variability in outcomes, and ultimately provide more effective, safer, and personalized treatment options for individuals with hemophilia.Item CAR- and TRuC-redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII(Elsevier, 2021) Rana, Jyoti; Perry, Daniel J.; Kumar, Sandeep R.P.; Muñoz-Melero, Maite; Saboungi, Rania; Brusko, Todd M.; Biswas, Moanaro; Pediatrics, School of MedicineRegulatory T cells (Tregs) control immune responses in autoimmune disease, transplantation, and enable antigen-specific tolerance induction in protein-replacement therapies. Tregs can exert a broad array of suppressive functions through their T cell receptor (TCR) in a tissue-directed and antigen-specific manner. This capacity can now be harnessed for tolerance induction by "redirecting" polyclonal Tregs to overcome low inherent precursor frequencies and simultaneously augment suppressive functions. With the use of hemophilia A as a model, we sought to engineer antigen-specific Tregs to suppress antibody formation against the soluble therapeutic protein factor (F)VIII in a major histocompatibility complex (MHC)-independent fashion. Surprisingly, high-affinity chimeric antigen receptor (CAR)-Treg engagement induced a robust effector phenotype that was distinct from the activation signature observed for endogenous thymic Tregs, which resulted in the loss of suppressive activity. Targeted mutations in the CD3ζ or CD28 signaling motifs or interleukin (IL)-10 overexpression were not sufficient to restore tolerance. In contrast, complexing TCR-based signaling with single-chain variable fragment (scFv) recognition to generate TCR fusion construct (TRuC)-Tregs delivered controlled antigen-specific signaling via engagement of the entire TCR complex, thereby directing functional suppression of the FVIII-specific antibody response. These data suggest that cellular therapies employing engineered receptor Tregs will require regulation of activation thresholds to maintain optimal suppressive function.Item Coagulation factor IX gene transfer to non-human primates using engineered AAV3 capsid and hepatic optimized expression cassette(Elsevier, 2021-08-26) Kumar, Sandeep R. P.; Xie, Jun; Hu, Shilang; Ko, Jihye; Huang, Qifeng; Brown, Harrison C.; Srivastava, Alok; Markusic, David M.; Doering, Christopher B.; Spencer, H. Trent; Srivastava, Arun; Gao, Guangping; Herzog, Roland W.; Pediatrics, School of MedicineHepatic gene transfer with adeno-associated viral (AAV) vectors shows much promise for the treatment of the X-linked bleeding disorder hemophilia B in multiple clinical trials. In an effort to further innovate this approach and to introduce alternative vector designs with potentially superior features into clinical development, we recently built a vector platform based on AAV serotype 3 because of its superior tropism for human hepatocytes. A vector genome with serotype-matched inverted terminal repeats expressing hyperactive human coagulation factor IX (FIX)-Padua was designed for clinical use that is optimized for translation using hepatocyte-specific codon-usage bias and is depleted of immune stimulatory CpG motifs. Here, this vector genome was packaged into AAV3 (T492V + S663V) capsid for hepatic gene transfer in non-human primates. FIX activity within or near the normal range was obtained at a low vector dose of 5 × 1011 vector genomes/kg. Pre-existing neutralizing antibodies, however, completely or partially blocked hepatic gene transfer at that dose. No CD8+ T cell response against capsid was observed. Antibodies against the human FIX transgene product formed at a 10-fold higher vector dose, albeit hepatic gene transfer was remarkably consistent, and sustained FIX activity in the normal range was nonetheless achieved in two of three animals for the 3-month duration of the study. These results support the use of this vector at low vector doses for gene therapy of hemophilia B in humans.Item Effect of CpG Depletion of Vector Genome on CD8+ T Cell Responses in AAV Gene Therapy(Frontiers Media, 2021-05-31) Bertolini, Thais B.; Shirley, Jamie L.; Zolotukhin, Irene; Li, Xin; Kaisho, Tsuneyasu; Xiao, Weidong; Kumar, Sandeep R.P.; Herzog, Roland W.; Pediatrics, School of MedicineAdeno associated viral (AAV) vectors have emerged as a preferred platform for in vivo gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product. In particular, TLR9 recognition of the vector's DNA genome in plasmacytoid dendritic cells (pDCs) has been identified as a key factor. Data from clinical trials and pre-clinical studies implement CpG motifs in the vector genome as drivers of immune responses, especially of CD8+ T cell activation. Here, we demonstrate that cross-priming of AAV capsid-specific CD8+ T cells depends on XCR1+ dendritic cells (which are likely the main cross-presenting cell that cooperates with pDCs to activate CD8+ T cells) and can be minimized by the elimination of CpG motifs in the vector genome. Further, a CpG-depleted vector expressing human coagulation factor IX showed markedly reduced (albeit not entirely eliminated) CD8+ T cell infiltration upon intramuscular gene transfer in hemophilia B mice when compared to conventional CpG+ vector (comprised of native sequences), resulting in better preservation of transduced muscle fibers. Therefore, this deimmunization strategy is helpful in reducing the potential for CD8+ T cell responses to capsid or transgene product. However, CpG depletion had minimal effects on antibody responses against capsid or transgene product, which appear to be largely independent of CpG motifs.Item Potential Role for Oral Tolerance in Gene Therapy(Elsevier, 2023) Butterfield, John S. S.; Li, Xin; Arisa, Sreevani; Kwon, Kwang-Chul; Daniell, Henry; Herzog, Roland W.; Pediatrics, School of MedicineOral immunotherapies are being developed for various autoimmune diseases and allergies to suppress immune responses in an antigen-specific manner. Previous studies have shown that anti-drug antibody (inhibitor) formation in protein replacement therapy for the inherited bleeding disorder hemophilia can be prevented by repeated oral delivery of coagulation factor antigens bioencapsulated in transplastomic lettuce cells. Here, we find that this approach substantially reduces antibody development against factor VIII in hemophilia A mice treated with adeno-associated viral gene transfer. We propose that the concept of oral tolerance can be applied to prevent immune responses against therapeutic transgene products expressed in gene therapy.Item Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia(Frontiers, 2020-05) Kumar, Sandeep R. P.; Wang, Xiaomei; Avuthu, Nagavardhini; Bertolini, Thais B.; Terhorst, Cox; Guda, Chittibabu; Daniell, Henry; Herzog, Roland W.; Pediatrics, School of MedicineFusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25-FoxP3-LAP+ T cells in the lamina propria of the small but not large intestine. By contrast, no changes in frequencies of CD4+CD25+FoxP3+ T cells were observed. Here we demonstrate that, surprisingly, the adoptive transfer of very small numbers of CD4+CD25-LAP+ Treg isolated from the spleen of tolerized mice significantly suppress antibodies directed against FIX. By contrast, equal numbers of splenic CD4+CD25+ T cells do not have an effect on antibody formation. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine and that suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg. The observation that CD4+CD25-LAP+ Treg migrate to the spleen are useful for the design of clinical protocols.Item Ryan White: A Geospatial Analysis of his Correspondence(2020-05) Shaeffer, Haley Lynn; Johnson, Daniel; Wilson, Jeffrey; Lulla, VijayThe letters Ryan White received over the course of his diagnosis, illness, and eventual death show a spatial distribution that reflected the United States’ response to Ryan’s condition. Ryan was diagnosed with AIDS in December of 1984 at the height of the epidemic, and the panic that surrounded it. In 2000, the Children’s Museum of Indianapolis accessioned a selection of letters sent to Ryan White and his mother, from 1980 to 1993. The expanded incorporation of these letters into the museum’s “Power of Children” gallery will introduce museum visitors to the public view on Ryan and the role he played in developing the public perception and awareness of AIDS in the 1980’s. Originally, it was anticipated that the distribution and number of letters Ryan received directly related to the concentration and spread of AIDS cases around the US. This research assumed that the AIDS community would have been more supportive and empathetic of Ryan’s diagnosis, resulting in those populations sending a higher number of letters. This assumption was also informed by the fact that the highest number of AIDS cases were in areas with large populations such as New York City, Los Angeles, and Miami. Yet findings showed relatively few letters were coming from the populated coasts where AIDS was more prevalent, and many more letters than expected came from areas with lower populations across the US. Ryan was one of the first children to go public with his AIDS diagnosis, which sparked strong reactions among people throughout the United States. Ryan’s correspondence and the outpouring of support he received allows insight into the multifaceted reaction to the AIDS crisis, especially from young people. Before Ryan became associated with the AIDS epidemic, this disease was seen primarily as an urban, gay, and drug-user related issue. The goal of this research is to gain further understanding of society’s shifting response to Ryan and AIDS during the 1980’s, by placing these letters in their social and geographic context.Item Ultrasound-mediated gene delivery of factor VIII plasmids for hemophilia A gene therapy in mice(Elsevier, 2022-01-10) Song, Shuxian; Lyle, Meghan J.; Noble-Vranish, Misty L.; Min-Tran, Dominic M.; Harrang, James; Xiao, Weidong; Unger, Evan C.; Miao, Carol H.; Pediatrics, School of MedicineGene therapy offers great promises for a cure of hemophilia A resulting from factor VIII (FVIII) gene deficiency. We have developed and optimized a non-viral ultrasound-mediated gene delivery (UMGD) strategy. UMGD of reporter plasmids targeting mice livers achieved high levels of transgene expression predominantly in hepatocytes. Following UMGD of a plasmid encoding human FVIII driven by a hepatocyte-specific promoter/enhancer (pHP-hF8/N6) into the livers of hemophilia A mice, a partial phenotypic correction was achieved in treated mice. In order to achieve persistent and therapeutic FVIII gene expression, we adopted a plasmid (pHP-hF8-X10) encoding an FVIII variant with significantly increased FVIII secretion. By employing an optimized pulse-train ultrasound condition and immunomodulation, the treated hemophilia A mice achieved 25%–150% of FVIII gene expression on days 1–7 with very mild transient liver damage, as indicated by a small increase of transaminase levels that returned to normal within 3 days. Therapeutic levels of FVIII can be maintained persistently without the generation of inhibitors in mice. These results indicate that UMGD can significantly enhance the efficiency of plasmid DNA transfer into the liver. They also demonstrate the potential of this novel technology to safely and effectively treat hemophilia A.