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Hematology ◽  
2021 ◽  
Vol 2021 (1) ◽  
pp. 226-233
Author(s):  
Lindsey A. George

Abstract After 3 decades of clinical trials, repeated proof-of-concept success has now been demonstrated in hemophilia A and B gene therapy. Current clinical hemophilia gene therapy efforts are largely focused on the use of systemically administered recombinant adeno-associated viral (rAAV) vectors for F8 or F9 gene addition. With multiple ongoing trials, including licensing studies in hemophilia A and B, many are cautiously optimistic that the first AAV vectors will obtain regulatory approval within approximately 1 year. While supported optimism suggests that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized, a number of outstanding questions have emerged from clinical trial that are in need of answers to harness the full potential of gene therapy for hemophilia patients. This article reviews the use of AAV vector gene addition approaches for hemophilia A and B, focusing specifically on information to review in the process of obtaining informed consent for hemophilia patients prior to clinical trial enrollment or administering a licensed AAV vector.


Author(s):  
Seung-Chan Kim ◽  
Ajung Kim ◽  
Jae-Yong Park ◽  
Eun Mi Hwang

2021 ◽  
Author(s):  
◽  
Hanane Belhoul-Fakir

<p>Activated microglia promote central nervous system (CNS) inflammation through antigen presentation and secretion of pro-inflammatory cytokines and chemokines. Although this activation is necessary to protect the brain during infection, aberrant release of pro-inflammatory and/or cytotoxic factors such as tumor necrosis factor-α, interleukin-1β, nitric oxide and reactive oxygen substances may lead to neuronal damage and degeneration.  Targeting microglia during neuroinflammation to regulate the expression of cytokines without affecting other cell types in the CNS is challenging since no specific microglial markers have yet been established that distinguish microglia from infiltrating, peripheral myeloid cells. Therefore, we propose that a viral-based gene delivery system might be a better strategy to regulate gene expression in microglia. Using the recombinant Adeno-associated virus (AAV) vector pseudotype 2/5, which preferentially infects microglia (, we constructed a plasmid backbone which contains GFP under the control of the F4/80 promoter, a macrophage-specific marker. In order to demonstrate the specificity of this promoter for macrophages, we transfected human kidney cells HEK 293 cells, mouse leukemic macrophages RAW 264.7 cells, human hepatocytes cell line (HepG2) and human ovarian carcinoma cell line (1A9) with the AAV-F4/80-eGFP construct or the control plasmid AAV-CAG-eGFP. Our results indicate that the rAAV-F4/80-GFP construct is selective for macrophages.  To begin to assess the usefulness of this system to alter microglia function, we have cloned the Membrane Associated Ring-CH protein (MARCHI) into the rAAV-F4/80-eGFP vector that has been shown earlier to regulate antigen presentation by inducing the intracellular sequestration of MHC class II. We were able to confirm this finding by transfecting interferon gamma stimulated macrophages cell line RAW 264.7 cells via our constructed AAV-F4/80-MARCHI-eGFP vector and demonstrate the ability of our recombinant AAV vector that is driven by specific promoter to deliver and express MARCHI to induce MHC class II sequestration. Together this work will lead to the development of tools that will allow us to dissect the pathways by which microglia promote neuroinflammation.</p>


2021 ◽  
Author(s):  
◽  
Hanane Belhoul-Fakir

<p>Activated microglia promote central nervous system (CNS) inflammation through antigen presentation and secretion of pro-inflammatory cytokines and chemokines. Although this activation is necessary to protect the brain during infection, aberrant release of pro-inflammatory and/or cytotoxic factors such as tumor necrosis factor-α, interleukin-1β, nitric oxide and reactive oxygen substances may lead to neuronal damage and degeneration.  Targeting microglia during neuroinflammation to regulate the expression of cytokines without affecting other cell types in the CNS is challenging since no specific microglial markers have yet been established that distinguish microglia from infiltrating, peripheral myeloid cells. Therefore, we propose that a viral-based gene delivery system might be a better strategy to regulate gene expression in microglia. Using the recombinant Adeno-associated virus (AAV) vector pseudotype 2/5, which preferentially infects microglia (, we constructed a plasmid backbone which contains GFP under the control of the F4/80 promoter, a macrophage-specific marker. In order to demonstrate the specificity of this promoter for macrophages, we transfected human kidney cells HEK 293 cells, mouse leukemic macrophages RAW 264.7 cells, human hepatocytes cell line (HepG2) and human ovarian carcinoma cell line (1A9) with the AAV-F4/80-eGFP construct or the control plasmid AAV-CAG-eGFP. Our results indicate that the rAAV-F4/80-GFP construct is selective for macrophages.  To begin to assess the usefulness of this system to alter microglia function, we have cloned the Membrane Associated Ring-CH protein (MARCHI) into the rAAV-F4/80-eGFP vector that has been shown earlier to regulate antigen presentation by inducing the intracellular sequestration of MHC class II. We were able to confirm this finding by transfecting interferon gamma stimulated macrophages cell line RAW 264.7 cells via our constructed AAV-F4/80-MARCHI-eGFP vector and demonstrate the ability of our recombinant AAV vector that is driven by specific promoter to deliver and express MARCHI to induce MHC class II sequestration. Together this work will lead to the development of tools that will allow us to dissect the pathways by which microglia promote neuroinflammation.</p>


Gene Therapy ◽  
2021 ◽  
Author(s):  
Winston Colon-Moran ◽  
Alan Baer ◽  
Gauri Lamture ◽  
Jack T. Stapleton ◽  
Joseph W. Fischer ◽  
...  

AbstractViral vector-mediated gene therapies have the potential to treat many human diseases; however, host immune responses against the vector and/or the transgene pose a safety risk to the patients and can negatively impact product efficacy. Thus, novel strategies to reduce vector immunogenicity are critical for the advancement of these therapies. T cell activation (TCA) is required for the development of immune responses during gene therapy. We hypothesized that modulation of TCA by incorporating a novel viral immunomodulatory factor into a viral vector may reduce unwanted TCA and immune responses during gene therapy. To test this hypothesis, we identified an immunomodulatory domain of the hepatitis C virus (HCV) NS protein 5A (NS5A) protein and studied the effect of viral vectors expressing NS5A peptide on TCA. Lentiviral vector-mediated expression of a short 20-mer peptide derived from the NS5A protein in human T cells was sufficient to inhibit TCA. Synthetic 20-mer NS5A peptide also inhibited TCA in primary human T cells. Mechanistically, the NS5A protein interacted with Lck and inhibited proximal TCR signaling. Importantly, NS5A peptide expression did not cause global T cell signaling dysfunction as distal T cell signaling was not inhibited. Finally, recombinant adeno-associated virus (AAV) vector expressing the 20-mer NS5A peptide reduced both the recall antigen and the TCR-mediated activation of human T cells and did not cause global T cell signaling dysfunction. Together, these data suggest that expression of a 20-mer NS5A peptide by an AAV vector may reduce unwanted TCA and may contribute to lower vector immunogenicity during gene therapy.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1853-1853
Author(s):  
Klaudia Kuranda ◽  
Christine Weisshaar ◽  
Yifeng Chen ◽  
Corinne Smith ◽  
Heena Beck ◽  
...  

Abstract Hemophilia is an X-linked bleeding disorder typically resulting in deficiency of factor VIII (FVIII) or factor IX (FIX) due to mutations in the F8 or F9 genes, respectively. Data from clinical trials have shown that the investigational delivery of functional F8 or F9 gene by recombinant adeno-associated virus (AAV) to hepatocytes can substantially eliminate the need for infusions of clotting factor in hemophilia patients. Despite major advances, the durability and redosing of these investigational gene therapies have been limited by the host immune response against the AAV capsid (Verdera et al. Mol Ther 2020). Currently, an oral corticosteroid, prednisone, is commonly used to prevent cytotoxic T cells from killing AAV-transduced hepatocytes and to sustain the production of transgenic clotting factor. However, in some instances, transgene expression was lost despite prednisone administration and prolonged use of prednisone can be associated with adverse effects. Previously, we demonstrated that the anti-capsid humoral response depends on interleukin 6 (IL-6) secretion from human monocyte-related dendritic cells (Kuranda et al. JCI 2018). IL-6 signaling in response to AAV was also observed in human non-parenchymal liver cells in vitro, animal gene transfer models and AAV-based gene therapy trial for hemophilia B (Konkle et al. Blood 2021). Here, we investigated the effects of the IL-6 signaling blockade as a possible targeted approach to modulate AAV vector immunogenicity in hemophilia gene therapy using a non-human primate (NHP) model. Spk100 AAV capsid was used to deliver the human F9 gene. To prevent IL-6 signaling, we used a monoclonal antibody, tocilizumab (TCZ), which blocks the IL-6 receptor. TCZ is currently approved for use in several forms of arthritis and cytokine release syndrome. Ten male cynomolgus monkeys received an intravenous injection of Spk100-hFIX vector (4x10 12 vg/kg) and 5 of those animals received a single dose of TCZ (8 mg/kg) the day prior to vector administration and were monitored for 13 weeks. As assessed by an array of clinical and anatomic pathology parameters, the investigational use of gene therapy combined with prophylactic TCZ administration was safe and well-tolerated. TCZ did not interfere with vector biodistribution, liver transduction or the transgenic FIX production. Spk100-hFIX alone modestly increased IL-6 secretion from NHP peripheral blood mononuclear cells (PBMC) in vitro but, following the vector infusion in animals, plasma IL-6 levels did not change significantly. Overall, cytokine secretion in response to Spk100 capsid and hFIX protein was lower in PBMCs isolated from TCZ-treated animals compared to cells obtained from control animals. As expected, animals administered Spk100-hFIX alone developed anti-capsid antibodies. The administration of TCZ was associated with a lower level of anti-Spk100 IgM, IgG and neutralizing antibodies (NAb). While the blockade of IL-6 signaling was effective for about for 14 days post-vector infusion, the lower level of anti-Spk100 antibodies persisted for the entire study duration. In the TCZ group, 4 out of 5 animals had NAb titers equal to or below 1:10, theoretically compatible with vector readministration. In contrast, 4 out of 5 animals in the control group developed high titer NAbs following vector infusion. Importantly, TCZ reduced the detection of the capsid-specific TNFα-positive T cells that were observed in animals after vector infusion. Finally, livers from sacrificed animals were used to prepare liver non-parenchymal cells for ex vivo phenotyping. Compared to the control animals, liver-resident immune cells from the TCZ-treated group had increased basal IL-10 secretion, while liver sinusoidal endothelial cells (albumin-CD45-CD31+CD146+MHCII+) had lower surface levels of MHC class II molecules, suggesting an anti-inflammatory milieu in the TCZ-treated livers. Our results show that short-term prophylactic blockade of IL-6 signaling was safely used in NHPs and has the potential to reduce immune responses commonly observed post AAV vector infusion. These results support the continued investigation of tocilizumab as a targeted immunomodulatory regimen in liver gene therapy for hemophilia. Disclosures Kuranda: Spark Therapeutics: Current Employment. Weisshaar: Spark Therapeutics: Current Employment. Chen: Spark Therapeutics: Ended employment in the past 24 months. Smith: Spark Therapeutics: Current Employment. Beck: Spark Therapeutics: Current Employment. Kahle: Spark Therapeutics: Current Employment. Mingozzi: Spark Therapeutics: Current Employment.


2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi163-vi163
Author(s):  
Dan Laks ◽  
Kenny Chen ◽  
Xiaoqin Ren ◽  
Ishan Shah ◽  
Usman Hameedi ◽  
...  

Abstract BACKGROUND HER2+ tumors constitute approximately 20% of breast cancer patients and are characterized by overexpression of the growth factor receptor HER2 (ERBB2), a cell proliferation driver. Effective anti-HER2 therapies confer prolonged patient survival necessitating the need for transformative treatments targeting brain metastases, a major cause of mortality in ~30-50% of HER2+ metastatic breast cancer patients. HER2-directed antibody immunotherapy, while efficacious for peripheral disease, has limited central nervous system exposure (CNS). To overcome these challenges, we transduced CNS cells with a novel AAV vector carrying an anti-HER2 antibody payload. METHODS We assessed the biochemical equivalence and functional effectiveness of AAV vector-encoded antibodies using in vitro assays. After selecting promising vector-encoded antibody candidates, a novel, blood-brain barrier penetrant AAV capsid was administered via i.v. dosing to an orthotopic xenograft mouse model of HER2+ brain metastases. Bioluminescent imaging provided a longitudinal measure of brain tumor burden. At study termination, we measured antibody biodistribution in cerebrospinal fluid (CSF), serum, and brain homogenates with AlphaLISA assays. RESULTS Using HER2+ breast cancer cell lines, we determined that an antibody-dependent cell cytotoxicity (ADCC) enhanced anti-HER2 antibody was most effective and demonstrated that AAV-vector encoded forms of the antibody performed comparably to recombinant reference antibodies. Following i.v. administration of a HER2 antibody encoding AAV vector, we measured &gt;1 ug/mL of the antibody in CSF. Importantly, AAV-mediated expression of the ADCC-enhanced HER2-directed antibody significantly abrogated tumor growth in orthotopic xenograft models. CONCLUSIONS Peripheral administration of an AAV vector was able to transduce brain tissue such that efficacious levels of HER2-directed antibodies were produced. This strategy was successful at preventing tumor growth in our physiologically relevant model of breast cancer brain metastases. Such a treatment modality should be further evaluated in patient derived PDX models to validate translational efficacy for human patients.


2021 ◽  
Vol 7 (9) ◽  
pp. 1183-1194
Author(s):  
Rachel, Legmann ◽  
Joe Ferraiolo

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