ABSTRACT
Accurately quantifying the functional consequences of non-coding mosaic variants requires the pairing of DNA sequence with both accessible and closed chromatin architectures along individual DNA molecules-a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic and epigenomic profiling across targeted >100 kilobase loci with ~10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the DMPK CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent. Furthermore, we reveal that therapeutic adenine base editing of the segmentally duplicated γ-globin (HBG1/HBG2) promoters in primary human hematopoietic cells induced towards an erythroblast lineage increases the accessibility of the HBG1 promoter as well as neighboring regulatory elements. Overall, we find that these non-protein coding mosaic variants can have complex impacts on chromatin architectures, including extending beyond the regulatory element harboring the variant.
ABSTRACT
ABSTRACT: We have reported the direct repair of the sickle cell mutation in vivo in a disease model using vectorized prime editors after hematopoietic stem cell (HSC) mobilization with granulocyte colony-stimulating factor (G-CSF)/AMD3100. The use of G-CSF for HSC mobilization is a hurdle for the clinical translation of this approach. Here, we tested a G-CSF-free mobilization regimen using WU-106, an inhibitor of integrin α4ß1, plus AMD3100 for in vivo HSC prime editing in sickle cell disease (SCD) mice. Mobilization with WU-106 + AMD3100 in SCD mice was rapid and efficient. In contrast to the G-CSF/AMD3100 approach, mobilization of activated granulocytes and elevation of the key proinflammatory cytokine interleukin-6 in the serum were minimal. The combination of WU-106 + AMD3100 mobilization and IV injection of the prime editing vector together with in vivo selection resulted in â¼23% correction of the SCD mutation in the bone marrow and peripheral blood cells of SCD mice. The treated mice demonstrated phenotypic correction, as reflected by normalized blood parameters and spleen size. Editing frequencies were significantly increased (29%) in secondary recipients, indicating the preferential mobilization/transduction of long-term repopulating HSCs. Using this approach, we found <1% undesired insertions/deletions and no detectable off-target editing at the top-scored potential sites. Our study shows that in vivo transduction to treat SCD can now be done within 2 hours involving only simple IV injections with a good safety profile. The same-day mobilization regimen makes in vivo HSC gene therapy more attractive for resource-poor settings, where SCD does the most damage.
Subject(s)
Anemia, Sickle Cell , Genetic Therapy , Granulocyte Colony-Stimulating Factor , Hematopoietic Stem Cell Mobilization , Animals , Humans , Mice , Anemia, Sickle Cell/therapy , Anemia, Sickle Cell/genetics , Benzylamines , Cyclams/pharmacology , Cyclams/therapeutic use , Disease Models, Animal , Gene Editing , Genetic Therapy/methods , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/metabolism , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/therapeutic useABSTRACT
Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein, Ad35K++, which is capable of transiently removing CD46 from the cell surface. We also produced a peptide inhibitor of CD59 (rILYd4). In this study, we tested Ad35K++ and rILYd4 in combination with daratumumab and isatuximab in MM cells as well as in cells from two other B-cell malignancies. We showed that Ad35K++ and rILYd4 increased CDC triggered by daratumumab and isatuximab. The combination of both inhibitors had an additive effect in vitro in primary MM cells as well as in vivo in a mouse xenograft model of MM. Daratumumab and isatuximab treatment of MM lines (without Ad35K++ or rILYd4) resulted in the upregulation of CD46/CD59 and/or survival of CD46high/CD59high MM cells that escaped the second round of daratumumab and isatuximab treatment. The escape in the second treatment cycle was prevented by the pretreatment of cells with Ad35K++. Overall, our data demonstrate that Ad35K++ and rILYd4 are efficient co-therapeutics of daratumumab and isatuximab, specifically in multi-cycle treatment regimens, and could be used to improve treatment of multiple myeloma.
Subject(s)
Antineoplastic Agents , Multiple Myeloma , Humans , Mice , Animals , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Agents/therapeutic use , ADP-ribosyl Cyclase 1/metabolism , CD59 Antigens/therapeutic use , Membrane Cofactor Protein/metabolismABSTRACT
In previous studies, we achieved safe and efficient in vivo hematopoietic stem cell (HSC) transduction in mobilized mice and macaques with intravenously injected helper-dependent adenovirus HDAd5/35++ vectors. These vectors are derivatives of serotype Ad5-containing CD46-affinity enhanced Ad35 fiber knob domains. Considering the impact of anti-Ad5/HDAd5/35++ neutralizing serum antibodies present in the human population, we generated HSC-retargeted HDAd6/35++ vectors derived from serotype 6. We found a lower prevalence and titers of serum anti-HDAd6/35++ in human samples compared with HDAd5/35++. HDAd6/35++ vectors efficiently transduced human and rhesus CD34+ cells in vitro. Intravenous injection of HDAd5/35++-GFP or HDAd6/35++-GFP vectors after G-CSF/AMD3100 mobilization of mice with established human hematopoiesis or human CD46 transgenic mice resulted in comparable GFP marking rates in HSCs in the bone marrow and spleen. In long-term in vivo HSC transduction and selection studies with integrating vectors, stable GFP expression in >75% of PBMCs was show for both vectors. In contrast with HDAd5/35++, undesired transduction of hepatocytes was minimal with HDAd6/35++. Furthermore, HDAd6/35++ allowed for efficient in vivo HSC transduction in Ad5-pre-immune mice. These features, together with the straightforward production of HDAd6/35++ vectors at high yield, make this new HDAd vector platform attractive for clinical translation of the in vivo approach.
ABSTRACT
We aim to develop an in vivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of â¼100 µg/mL (mice) and >20 µg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1 µg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were â¼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+ cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the in vivo approach. Clearly, our conclusions need to be validated in larger animal cohorts.
Subject(s)
HIV Infections , HIV-1 , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Humans , Animals , Mice , Macaca mulatta , Simian Immunodeficiency Virus/genetics , Hematopoietic Stem Cells , Simian Acquired Immunodeficiency Syndrome/therapyABSTRACT
Sickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the ß-globin gene. Current gene therapy studies are mainly focused on lentiviral vector-mediated gene addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a nonintegrating, prime editor-expressing viral vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of â¼40% of ßS alleles in HSCs. On average, 43% of sickle hemoglobin was replaced by adult hemoglobin, thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.
Subject(s)
Anemia, Sickle Cell , Gene Editing , Mice , Animals , Gene Editing/methods , CRISPR-Cas Systems , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/therapy , Hematopoietic Stem Cells , Hemoglobin, Sickle/geneticsABSTRACT
INTRODUCTION: Prompt and effective hemorrhage control is paramount to improve survival in patients with catastrophic bleeding. In the ever-expanding field of bleeding control techniques, there is a need for a realistic training model to practice these life-saving skills. This study aimed to create a realistic perfused post-mortem human specimen (PMHS) flow model that is suitable for training various bleeding control techniques. MATERIALS AND METHODS: This laboratory study was conducted in the SkillsLab & Simulation Center of Erasmus MC, University Medical Center Rotterdam, the Netherlands. One fresh frozen and five AnubiFiX® embalmed PMHS were used for the development of the model. Subsequent improvements in the exact preparation and design of the flow model were made based on model performance and challenges that occurred during this study and are described. RESULTS: Circulating arteriovenous flow with hypertonic saline was established throughout the entire body via inflow and outflow cannulas in the carotid artery and jugular vein of embalmed PMHS. We observed full circulation and major hemorrhage could be mimicked. Effective bleeding control was achieved by placing a resuscitative endovascular balloon occlusion of the aorta (REBOA) catheter in the model. Regional perfusion significantly reduced the development of tissue edema. CONCLUSION: Our perfused PMHS model with circulating arterial and venous flow appears to be a feasible method for the training of multiple bleeding control techniques. Regional arteriovenous flow successfully reduces tissue edema and increases the durability of the model. Further research should focus on reducing edema and enhancing the durability of the model.
Subject(s)
Balloon Occlusion , Endovascular Procedures , Humans , Balloon Occlusion/methods , Cadaver , Hemorrhage/therapy , Aorta , Netherlands , Resuscitation/methodsABSTRACT
Desmoglein 2 (DSG2) is overexpressed in many epithelial cancers and therefore represents a target receptor for oncolytic viruses, including Ad5/3-based viruses. For most Ad serotypes, the receptor-binding fiber is composed of tail, shaft, and knob domains. Here, we investigated the role of the fiber shaft in Ad5/3 tumor transduction in vitro and in human DSG2-transgenic mice carrying human DSG2high tumors. DSG2tg mice express DSG2 in a pattern similar to humans. We constructed Ad5/3L (with the "long" Ad5 shaft) and Ad5/3S (with the "short" Ad3 shaft) expressing GFP or luciferase. In in vitro studies we found that coagulation factor X, which is known to mediate undesired hepatocyte transduction of Ad5, enhances the transduction of Ad5/3(L), but not the transduction of Ad5/3(S). We therefore hypothesized that Ad5/3(S) would target DSG2high tumors while sparing the liver after intravenous injection. In vivo imaging studies for luciferase and analysis of luciferase activity in isolated organs, showed that Ad5/3(L) vectors efficiently transduced DSG2high tumors and liver but not normal epithelial tissues after intravenous injection. Ad5/3(S) showed minimal liver transduction, however it failed to transduce DSG2high tumors. Further modifications of the Ad5/3(S) capsid are required to compensate for the lower infectivity of Ad5/3(S) vectors.
Subject(s)
Adenoviruses, Human , Neoplasms , Humans , Mice , Animals , Adenoviruses, Human/genetics , Genetic Vectors/genetics , Capsid Proteins/genetics , Neoplasms/genetics , Neoplasms/therapy , LuciferasesABSTRACT
Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening. Junction opener 4 (JO-4), a small recombinant protein derived from the Ad3 fiber knob, was previously developed with a higher affinity to DSG2. JO-4 protein has been proven to enhance the effects of antibody therapy and chemotherapy and is now considered for clinical trials. However, the effect of the JO4 mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4 mutation to various adenoviral vectors to explore their infection properties. In the current experimental settings and investigated cell lines, the JO4-containing vectors showed no enhanced transduction compared with their parental vectors in DSG2-high cell lines. Moreover, in DSG2-low cell lines, the JO4 vectors presented a rather weakened effect. Interestingly, DSG2-negative cell line MIA PaCa-2 even showed resistance to JO4 vector infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor: CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46-mediated sequestration, thereby achieving DSG2-specific transduction.
Subject(s)
Adenoviruses, Human , Genetic Vectors , Adenoviruses, Human/physiology , Cell Line , Desmoglein 2/genetics , Desmoglein 2/metabolism , Genetic Therapy , Genetic Vectors/genetics , Humans , Intercellular JunctionsABSTRACT
Individuals with ß-thalassemia or sickle cell disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% fetal hemoglobin (HbF) appear to be symptom free. Here, we used a nonintegrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113 A>G HPFH mutation in the γ-globin promoters in healthy CD46/ß-YAC mice carrying the human ß-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only s.c. and i.v. injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/ß-YAC mice resulted in > 60% -113 A>G conversion, with 30% γ-globin of ß-globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-Seq. In vitro, in HSCs from ß-thalassemia and patients with sickle cell disease, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.
Subject(s)
Anemia, Sickle Cell , Hemoglobinopathies , beta-Thalassemia , Adenine , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/therapy , Animals , CRISPR-Cas Systems , Fetal Hemoglobin/genetics , Fetal Hemoglobin/metabolism , Gene Editing/methods , Hemoglobinopathies/genetics , Hemoglobinopathies/therapy , Humans , Mice , beta-Globins/genetics , beta-Thalassemia/genetics , beta-Thalassemia/therapy , gamma-Globins/geneticsABSTRACT
[This corrects the article DOI: 10.1016/j.omtm.2021.12.003.].
ABSTRACT
We developed a new in vivo hematopoietic stem cell (HSC) gene therapy approach that involves only IV injections and does not require myeloablation/conditioning and HSC transplantation. In this approach, HSCs are mobilized from the bone marrow into the peripheral bloodstream and transduced with IV injected helper-dependent adenovirus (HDAd) vectors. A fraction of transduced HSCs returns to the bone marrow and persists there long term. Here, we report desmoglein 2 (DSG2) as a new receptor that can be used for in vivo HSC transduction. HDAd5/3+ vectors were developed that use DSG2 as a high-affinity attachment receptor, and in vivo HSC transduction and safety after IV injection of an HDAd5/3+ vector expressing green fluorescent protein (GFP) in granulocyte colony-stimulating factor/AMD3100 (plerixafor)-mobilized rhesus macaques were studied. Unlike previously used CD46-targeting HDAd5/35++ vectors, HDAd5/3+ virions were not sequestered by rhesus erythrocytes and therefore mediated â¼10-fold higher GFP marking rates in primitive HSCs (CD34+/CD45RA-/CD90+ cells) in the bone marrow at day 7 after vector injection. To further increase the return of in vivo transduced, mobilized HSCs to the bone marrow, we transiently expressed cxcr4 in mobilized HSCs from the HDAd5/3+ vector. In vivo transduction with an HDAd5/3+GFP/cxcr4 vector at a low dose of 0.4 × 1012 viral particles/kg resulted in up to 7% of GFP-positive CD34+/CD45RA-/CD90+ cells in the bone marrow. This transduction rate is a solid basis for in vivo base or prime editing in combination with natural or drug-induced expansion of edited HSCs. Furthermore, our study provides new insights into HSC biology and trafficking after mobilization in nonhuman primates.
Subject(s)
Hematopoietic Stem Cell Mobilization , Heterocyclic Compounds , Animals , Antigens, CD34/metabolism , Desmoglein 2/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cells/metabolism , Heterocyclic Compounds/pharmacology , Macaca mulatta/metabolismABSTRACT
Our goal is to overcome treatment resistance in ovarian cancer patients which occurs in most cases after an initial positive response to chemotherapy. A central resistance mechanism is the maintenance of desmoglein-2 (DSG2) positive tight junctions between malignant cells that prevents drug penetration into the tumor. We have generated JO4, a recombinant protein that binds to DSG2 resulting in the transient opening of junctions in epithelial tumors. Here we present studies toward the clinical translation of c-JO4 in combination with PEGylated liposomal doxorubicin/Doxil for ovarian cancer therapy. A manufacturing process for cGMP compliant production of JO4 was developed resulting in c-JO4. GLP toxicology studies using material from this process in DSG2 transgenic mice and cynomolgus macaques showed no treatment-related toxicities after intravenous injection at doses reaching 24 mg/kg. Multiple cycles of intravenous c-JO4 plus Doxil (four cycles, 4 weeks apart, simulating the treatment regimen in the clinical trial) elicited antibodies against c-JO4 that increased with each cycle and were accompanied by elevation of pro-inflammatory cytokines IL-6 and TNFα. Pretreatment with steroids and cyclophosphamide reduced anti-c-JO4 antibody response and blunted cytokine release. Our data indicate acceptable safety of our new treatment approach if immune reactions are monitored and counteracted with appropriate immune suppression.
Subject(s)
Ovarian Neoplasms , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cyclophosphamide/therapeutic use , Doxorubicin , Female , Humans , Mice , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Recombinant Proteins/therapeutic use , Technology , Tight Junctions/pathologyABSTRACT
Abstract Background Anti-desmoglein 1 and 3 autoantibodies justify acantholysis in pemphigus; however, the pathogenesis of anti-desmoglein 2 is hypothetical. Objective To compare the participation of desmogleins 1, 2 and 3 through the production of serum autoantibodies, and protein and gene expression in the skin/mucosa of patients with pemphigus foliaceus and pemphigus vulgaris. Methods The autoantibodies were titrated by ELISA in 202 samples of pemphigus foliaceus, 131 pemphigus vulgaris, 50 and 57 relatives of patients with pemphigus foliaceus and pemphigus vulgaris, respectively, and 114 controls. Protein and gene expressions were determined by immunohistochemistry and qPCR in the skin/mucosa of 3 patients with pemphigus foliaceus and 3 patients with pemphigus vulgaris. Results Higher titers of anti-desmoglein 2 (optical density) resulted in pemphigus foliaceus and pemphigus vulgaris, when compared to controls (0.166; 0.180; 0.102; respectively; p < 0.0001). There was a correlation between anti-desmoglein 2 and anti-desmoglein 1 titers in pemphigus foliaceus (r = 0.1680; p = 0.0206). There was no cross-reaction of anti-desmoglein 2 with desmoglein 1 and 3. Protein overexpression of desmoglein 2 was observed in intact and lesional skin of patients with pemphigus compared to the skin of controls. Internalization granules of desmoglein 1 and 3, but not of desmoglein 2, were observed in lesions of pemphigus foliaceus and pemphigus vulgaris, respectively. Gene overexpression of desmoglein 2 was observed in the mucosa. Study limitations Small sample size for the statistical analysis of protein and gene expression. Conclusion Autoantibodies against desmoglein 2 are not pathogenic in pemphigus; protein and gene overexpression of desmoglein 2 in the skin and mucosa may be involved in acantholysis repair.
ABSTRACT
We tested a new in vivo hematopoietic stem cell (HSC) transduction/selection approach in rhesus macaques using HSC-tropic, integrating, helper-dependent adenovirus vectors (HDAd5/35++) designed for the expression of human γ-globin in red blood cells (RBCs) to treat hemoglobinopathies. We show that HDAd5/35++ vectors preferentially transduce HSCs in vivo after intravenous injection into granulocyte colony-stimulating factor (G-CSF)/AMD3100-mobilized animals and that transduced cells return to the bone marrow and spleen. The approach was well tolerated, and the activation of proinflammatory cytokines that are usually associated with intravenous adenovirus vector injection was successfully blunted by pre-treatment with dexamethasone in combination with interleukin (IL)-1 and IL-6 receptor blockers. Using our MGMTP140K-based in vivo selection approach, γ-globin+ RBCs increased in all animals with levels up to 90%. After selection, the percentage of γ-globin+ RBCs declined, most likely due to an immune response against human transgene products. Our biodistribution data indicate that γ-globin+ RBCs in the periphery were mostly derived from mobilized HSCs that homed to the spleen. Integration site analysis revealed a polyclonal pattern and no genotoxicity related to transgene integrations. This is the first proof-of-concept study in nonhuman primates to show that in vivo HSC gene therapy could be feasible in humans without the need for high-dose chemotherapy conditioning and HSC transplantation.
ABSTRACT
While SARS-CoV2 vaccines have shown an unprecedented success, the ongoing emergence of new variants and necessity to adjust vaccines justify the development of alternative prophylaxis and therapy approaches. Hematopoietic stem cell (HSC) gene therapy using a secreted CoV2 decoy receptor protein (sACE2-Ig) would involve a one-time intervention resulting in long-term protection against airway infection, viremia, and extrapulmonary symptoms. We recently developed a technically simple and portable in vivo hematopoietic HSC transduction approach that involves HSC mobilization from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. Considering the abundance of erythrocytes, in this study, we directed sACE2-Ig expression to erythroid cells using strong ß-globin transcriptional regulatory elements. We performed in vivo HSC transduction of CD46-transgenic mice with an HDAd-sACE2-Ig vector. Serum sACE2-Ig levels reached 500-1,300 ng/mL after in vivo selection. At 22 weeks, we used genetically modified HSCs from these mice to substitute the hematopoietic system in human ACE2-transgenic mice, thus creating a model that is susceptible to SARS-CoV2 infection. Upon challenge with a lethal dose of CoV2 (WA-1), sACE2-Ig expressed from erythroid cells of test mice diminishes infection sequelae. Treated mice lost significantly less weight, had less viremia, and displayed reduced cytokine production and lung pathology. The second objective of this study was to assess the safety of in vivo HSC transduction and long-term sACE2-Ig expression in a rhesus macaque. With appropriate cytokine prophylaxis, intravenous injection of HDAd-sACE2-Ig into the mobilized animal was well tolerated. In vivo transduced HSCs preferentially localized to and survived in the spleen. sACE2-Ig expressed from erythroid cells did not affect erythropoiesis and the function of erythrocytes. While these pilot studies are promising, the antiviral efficacy of the approach has to be improved, for example, by using of decoy receptors with enhanced neutralizing capacity and/or expression of multiple antiviral effector proteins.
Subject(s)
COVID-19 , RNA, Viral , Animals , COVID-19/therapy , Cytokines/metabolism , Genetic Therapy/methods , Hematopoietic Stem Cells/metabolism , Macaca mulatta , Mice , Mice, Transgenic , RNA, Viral/metabolism , SARS-CoV-2/genetics , Viremia/metabolismABSTRACT
BACKGROUND: Anti-desmoglein 1 and 3 autoantibodies justify acantholysis in pemphigus; however, the pathogenesis of anti-desmoglein 2 is hypothetical. OBJECTIVE: To compare the participation of desmogleins 1, 2 and 3 through the production of serum autoantibodies, and protein and gene expression in the skin/mucosa of patients with pemphigus foliaceus and pemphigus vulgaris. METHODS: The autoantibodies were titrated by ELISA in 202 samples of pemphigus foliaceus, 131 pemphigus vulgaris, 50 and 57 relatives of patients with pemphigus foliaceus and pemphigus vulgaris, respectively, and 114 controls. Protein and gene expressions were determined by immunohistochemistry and qPCR in the skin/mucosa of 3 patients with pemphigus foliaceus and 3 patients with pemphigus vulgaris. RESULTS: Higher titers of anti-desmoglein 2 (optical density) resulted in pemphigus foliaceus and pemphigus vulgaris, when compared to controls (0.166; 0.180; 0.102; respectively; pâ¯<â¯0.0001). There was a correlation between anti-desmoglein 2 and anti-desmoglein 1 titers in pemphigus foliaceus (râ¯=â¯0.1680; pâ¯=â¯0.0206). There was no cross-reaction of anti-desmoglein 2 with desmoglein 1 and 3. Protein overexpression of desmoglein 2 was observed in intact and lesional skin of patients with pemphigus compared to the skin of controls. Internalization granules of desmoglein 1 and 3, but not of desmoglein 2, were observed in lesions of pemphigus foliaceus and pemphigus vulgaris, respectively. Gene overexpression of desmoglein 2 was observed in the mucosa. STUDY LIMITATIONS: Small sample size for the statistical analysis of protein and gene expression. CONCLUSION: Autoantibodies against desmoglein 2 are not pathogenic in pemphigus; protein and gene overexpression of desmoglein 2 in the skin and mucosa may be involved in acantholysis repair.
Subject(s)
Pemphigus , Acantholysis , Autoantibodies , Desmoglein 1 , Desmoglein 2 , Desmoglein 3 , HumansABSTRACT
Oncolytic adenoviruses (OAd) represent an attractive treatment option for cancer. Clinical efficacy of commonly utilized human adenovirus type 5 (Ad5)-based oncolytic viruses is limited by variable expression levels of the coxsackie- and adenovirus receptor (CAR) in tumor cells and high prevalence of neutralizing antibodies against human Ad5. However, previous studies have highlighted alternative human Ad types as promising candidates for oncolytic therapy. In this study, we generated novel OAds based on Ad1, -2, -5, and -6 derived from species C Ads. These OAds contain a 24-bp deletion in the early gene E1A for tumor selective replication and express the RNAi inhibitor P19. We examined these OAds for in vitro anticancer activity on various cancer cell lines derived from lung, colon, gynecologic, bone, and pancreatic carcinoma. In most surveyed cell lines, OAds based on Ad1, -2, and -6 demonstrated higher cell lysis capability compared with Ad5, suggesting enhanced oncolytic potential. Moreover, enhanced oncolytic activity was associated with P19 expression in a cell type-dependent manner. We further explored a A549 tumor xenograft mouse model to compare the novel OAds directly with Ad5 and H101, an oncolytic adenovirus used in clinical trials. These P19-containing OAds based on Ad1, -2, and -6 showed significantly decelerated tumor progression compared with H101, indicating better antitumor potency in vivo. Our studies provide a novel path for OAd development based on alternative Ad types with improved effectiveness by RNA interference suppression.