Publications by authors named "Robert L Kruse"

19 Publications

  • Page 1 of 1

A hemagglutination-based, semi-quantitative test for point-of-care determination of SARS-CoV-2 antibody levels.

J Clin Microbiol 2021 Sep 1:JCM0118621. Epub 2021 Sep 1.

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Serologic, point-of-care tests to detect antibodies against SARS-CoV-2 are an important tool in the COVID-19 pandemic. The majority of current point-of-care antibody tests developed for SARS-CoV-2 rely on lateral flow assays, but these do not offer quantitative information. To address this, we developed a novel antibody test leveraging hemagglutination, employing a dry card format currently used for typing ABO blood groups. 200 COVID-19 patient and 200 control plasma samples were reconstituted with O-negative RBCs to form whole blood and added to dried viral-antibody fusion protein, followed by a stirring step and a tilting step, 3-minute incubation, and a second tilting step. The sensitivity for the hemagglutination test, Euroimmun IgG ELISA test and RBD-based CoronaChek lateral flow assay was 87.0%, 86.5%, and 84.5%, respectively, using samples obtained from recovered COVID-19 individuals. Testing pre-pandemic samples, the hemagglutination test had a specificity of 95.5%, compared to 97.3% and 98.9% for the ELISA and CoronaChek, respectively. A distribution of agglutination strengths was observed in COVID-19 convalescent plasma samples, with the highest agglutination score (4) exhibiting significantly higher neutralizing antibody titers than weak positives (2) (p<0.0001). Strong agglutinations were observed within 1 minute of testing, and this shorter assay time also increased specificity to 98.5%. In conclusion, we developed a novel rapid, point-of-care RBC agglutination test for the detection of SARS-CoV-2 antibodies that can yield semi-quantitative information on neutralizing antibody titer in patients. The five-minute test may find use in determination of serostatus prior to vaccination, post-vaccination surveillance and travel screening.
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http://dx.doi.org/10.1128/JCM.01186-21DOI Listing
September 2021

Endoscopic-mediated, biliary hydrodynamic injection mediating clinically relevant levels of gene delivery in pig liver.

Gastrointest Endosc 2021 Jun 29. Epub 2021 Jun 29.

Division of Gastroenterology & Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Division of Gastroenterology & Hepatology, Department of Medicine, Mayo Clinic Florida, Jacksonville, Florida, USA.

Background And Aims: Gene therapy could provide curative therapies to many inherited monogenic liver diseases. Clinical trials have largely focused on adeno-associated viruses (AAVs) for liver gene delivery. These vectors, however, are limited by small packaging size, capsid immune responses, and inability to redose. As an alternative, nonviral, hydrodynamic injection through vascular routes can successfully deliver plasmid DNA (pDNA) into mouse liver but has achieved limited success in large animal models.

Methods: We explored hydrodynamic delivery of pDNA through the biliary system into the liver of pigs using ERCP and a power injector to supply hydrodynamic force. Human factor IX (hFIX), deficient in hemophilia B, was used as a model gene therapy.

Results: Biliary hydrodynamic injection was well tolerated without significant changes in vital signs, liver enzymes, hematology, or histology. No off-target pDNA delivery to other organs was detected by polymerase chain reaction. Immunohistochemistry revealed that 50.19% of the liver stained positive for hFIX after hydrodynamic injection at 5.5 mg pDNA, with every hepatic lobule in all liver lobes demonstrating hFIX expression. hFIX-positive hepatocytes were concentrated around the central vein, radiating outward across all 3 metabolic zones. Biliary hydrodynamic injection in pigs resulted in significantly higher transfection efficiency than mouse vascular hydrodynamic injection at matched pDNA per liver weight dose (32.7%-51.9% vs 18.9%, P < .0001).

Conclusions: Biliary hydrodynamic injection using ERCP can achieve higher transfection efficiency into hepatocytes compared with AAVs at magnitudes of less cost in a clinically relevant human-sized large animal. This technology may serve as a platform for gene therapy of human liver diseases.
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http://dx.doi.org/10.1016/j.gie.2021.06.016DOI Listing
June 2021

A hemagglutination-based, semi-quantitative test for point-of-care determination of SARS-CoV-2 antibody levels.

medRxiv 2021 May 4. Epub 2021 May 4.

Serologic, point-of-care tests to detect antibodies against SARS-CoV-2 are an important tool in the COVID-19 pandemic. The majority of current point-of-care antibody tests developed for SARS-CoV-2 rely on lateral flow assays, but these do not offer quantitative information. To address this, we developed a new method of COVID-19 antibody testing employing hemagglutination tested on a dry card, similar to that which is already available for rapid typing of ABO blood groups. A fusion protein linking red blood cells (RBCs) to the receptor-binding domain (RBD) of SARS-CoV-2 spike protein was placed on the card. 200 COVID-19 patient and 200 control plasma samples were reconstituted with O-negative RBCs to form whole blood and added to the dried protein, followed by a stirring step and a tilting step, 3-minute incubation, and a second tilting step. The sensitivity for the hemagglutination test, Euroimmun IgG ELISA test and RBD-based CoronaChek lateral flow assay was 87.0%, 86.5%, and 84.5%, respectively, using samples obtained from recovered COVID-19 individuals. Testing pre-pandemic samples, the hemagglutination test had a specificity of 95.5%, compared to 97.3% and 98.9% for the ELISA and CoronaChek, respectively. A distribution of agglutination strengths was observed in COVID-19 convalescent plasma samples, with the highest agglutination score (4) exhibiting significantly higher neutralizing antibody titers than weak positives (2) (p<0.0001). Strong agglutinations were observed within 1 minute of testing, and this shorter assay time also increased specificity to 98.5%. In conclusion, we developed a novel rapid, point-of-care RBC agglutination test for the detection of SARS-CoV-2 antibodies that can yield semi-quantitative information on neutralizing antibody titer in patients. The five-minute test may find use in determination of serostatus prior to vaccination, post-vaccination surveillance and travel screening.
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http://dx.doi.org/10.1101/2021.05.01.21256452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109202PMC
May 2021

Parameters of biliary hydrodynamic injection during endoscopic retrograde cholangio-pancreatography in pigs for applications in gene delivery.

PLoS One 2021 28;16(4):e0249931. Epub 2021 Apr 28.

Division of Gastroenterology & Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

The biliary system is routinely accessed for clinical purposes via endoscopic retrograde cholangiopancreatography (ERCP). We previously pioneered ERCP-mediated hydrodynamic injection in large animal models as an innovative gene delivery approach for monogenic liver diseases. However, the procedure poses potential safety concerns related mainly to liver or biliary tree injury. Here, we sought to further define biliary hydrodynamic injection parameters that are well-tolerated in a human-sized animal model. ERCP was performed in pigs, and hydrodynamic injection carried out using a novel protocol to reduce duct wall stress. Each pig was subjected to multiple repeated injections to expedite testing and judge tolerability. Different injection parameters (volume, flow rate) and injection port diameters were tested. Vital signs were monitored throughout the procedure, and liver enzyme panels were collected pre- and post-procedure. Pigs tolerated repeated biliary hydrodynamic injections with only occasional, mild, isolated elevation in aspartate aminotransferase (AST), which returned to normal levels within one day post-injection. All other liver tests remained unchanged. No upper limit of volume tolerance was reached, which suggests the biliary tree can readily transmit fluid into the vascular space. Flow rates up to 10 mL/sec were also tolerated with minimal disturbance to vital signs and no anatomic rupture of bile ducts. Measured intrabiliary pressure was up to 150 mmHg, and fluid-filled vesicles were induced in liver histology at high flow rates, mimicking the changes in histology observed in mouse liver after hydrodynamic tail vein injection. Overall, our investigations in a human-sized pig liver using standard clinical equipment suggest that ERCP-guided hydrodynamic injection will be safely tolerated in patients. Future investigations will interrogate if higher flow rates and pressure mediate higher DNA delivery efficiencies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249931PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081268PMC
September 2021

A rapid, point-of-care red blood cell agglutination assay detecting antibodies against SARS-CoV-2.

Biochem Biophys Res Commun 2021 05 15;553:165-171. Epub 2021 Mar 15.

Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address:

The COVID-19 pandemic has caused significant morbidity and mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess past infection, evaluate responses to vaccines in development, and determine individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which have not scaled to meet the demand of hundreds of millions of antibody tests so far. Herein, we present an alternative method of antibody testing that depends on one protein reagent being added to patient serum/plasma or whole blood with direct, visual readout. Two novel fusion proteins, RBD-2E8 and B6-CH1-RBD, were designed to bind red blood cells (RBCs) via a single-chain variable fragment (scFv), thereby displaying the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Mixing mammalian-derived RBD-2E8 and B6-CH1-RBD with convalescent COVID-19 patient serum and RBCs led to visible hemagglutination, indicating the presence of antibodies against SARS-CoV-2 RBD. B6-CH1-RBD made in bacteria was not as effective in inducing agglutination, indicating better recognition of RBD epitopes from mammalian cells. Given that our hemagglutination test uses methods routinely used in hospital clinical labs across the world for blood typing, we anticipate the test can be rapidly deployed at minimal cost. We anticipate our hemagglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959259PMC
May 2021

A hepatitis B virus transgenic mouse model with a conditional, recombinant, episomal genome.

JHEP Rep 2021 Apr 6;3(2):100252. Epub 2021 Feb 6.

Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, TX, USA.

Background & Aims: Development of new and more effective therapies against hepatitis B virus (HBV) is limited by the lack of suitable small animal models. The HBV transgenic mouse model containing an integrated overlength 1.3-mer construct has yielded crucial insights, but this model unfortunately lacks covalently closed circular DNA (cccDNA), the episomal HBV transcriptional template, and cannot be cured given that HBV is integrated in every cell.

Methods: To solve these 2 problems, we generated a novel transgenic mouse (HBV1.1X), which generates an excisable circular HBV genome using Cre/LoxP technology. This model possesses a HBV1.1-mer cassette knocked into the locus and is designed for stable expression of viral proteins from birth, like the current HBV transgenic mouse model, before genomic excision with the introduction of Cre recombinase.

Results: We demonstrated induction of recombinant cccDNA (rcccDNA) formation via viral or transgenic Cre expression in HBV1.1X mice, and the ability to regulate HBsAg and HBc expression with Cre in mice. Tamoxifen-inducible Cre could markedly downregulate baseline HBsAg levels from the integrated HBV genome. To demonstrate clearance of HBV from HBV1.1X mice, we administered adenovirus expressing Cre, which permanently and significantly reduced HBsAg and core antigen levels in the murine liver via rcccDNA excision and a subsequent immune response.

Conclusions: The HBV1.1X model is the first Cre-regulatable HBV transgenic mouse model and should be of value to mimic chronic HBV infection, with neonatal expression and tolerance of HBV antigens, and on-demand modulation of HBV expression.

Lay Summary: Hepatitis B virus (HBV) can only naturally infect humans and chimpanzees. Mouse models have been developed with the HBV genome integrated into mouse chromosomes, but this prevents mice from being cured. We developed a new transgenic mouse model that allows for HBV to be excised from mouse chromosomes to form a recombinant circular DNA molecule resembling the natural circular HBV genome. HBV expression could be reduced in these mice, enabling curative therapies to be tested in this new mouse model.
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http://dx.doi.org/10.1016/j.jhepr.2021.100252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940981PMC
April 2021

Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection.

Virus Res 2021 01 6;292:198224. Epub 2020 Nov 6.

Center for Cell and Gene Therapy, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA.

New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P < 0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.
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http://dx.doi.org/10.1016/j.virusres.2020.198224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779077PMC
January 2021

Cryoprecipitate Utilization Patterns Observed With a Required Prospective Approval Process vs Electronic Dosing Guidance.

Am J Clin Pathol 2020 08;154(3):362-368

Division of Transfusion Medicine, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD.

Objectives: We evaluated the impact of electronic medical record (EMR)-guided pooled cryoprecipitate dosing vs our previous practice of requiring transfusion medicine (TM) resident approval for every cryoprecipitate transfusion.

Methods: At our hospital, cryoprecipitate pooled from five donors is dosed for adult patients, while single-donor cryoprecipitate is dosed for pediatric patients (defined as patients <50 kg in weight). EMR-based dosing guidance replaced a previously required TM consultation when cryoprecipitate pools were ordered, but a consultation remained required for single-unit orders. Usage was defined as thawed cryoprecipitate; wastage was defined as cryoprecipitate that expired prior to transfusion.

Results: In the 6 months prior to intervention, 178 ± 13 doses of pooled cryoprecipitate were used per month vs 187 ± 15 doses after the intervention (P = .68). Wastage of pooled cryoprecipitate increased from 7.7% ± 1.5% to 12.7% ± 1.4% (P = .038). There was no change in wastage of pediatric cryoprecipitate doses during the study period. These trends remained unchanged for a full year postimplementation.

Conclusions: Electronic dosing guidance resulted in similar cryoprecipitate usage as TM auditing. Increased wastage may result from reduced TM oversight. Product wastage should be balanced against the possibility that real-time audits could delay a lifesaving therapy.
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http://dx.doi.org/10.1093/ajcp/aqaa042DOI Listing
August 2020

Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China.

Authors:
Robert L Kruse

F1000Res 2020 31;9:72. Epub 2020 Jan 31.

Department of Pathology, Johns Hopkins Hospital, Baltimore, Maryland, 21287, USA.

A novel coronavirus (2019-nCoV) originating in Wuhan, China presents a potential respiratory viral pandemic to the world population. Current efforts are focused on containment and quarantine of infected individuals. Ultimately, the outbreak could be controlled with a protective vaccine to prevent 2019-nCoV infection. While vaccine research should be pursued intensely, there exists today no therapy to treat 2019-nCoV upon infection, despite an urgent need to find options to help these patients and preclude potential death. Herein, I review the potential options to treat 2019-nCoV in patients, with an emphasis on the necessity for speed and timeliness in developing new and effective therapies in this outbreak. I consider the options of drug repurposing, developing neutralizing monoclonal antibody therapy, and an oligonucleotide strategy targeting the viral RNA genome, emphasizing the promise and pitfalls of these approaches. Finally, I advocate for the fastest strategy to develop a treatment now, which could be resistant to any mutations the virus may have in the future. The proposal is a biologic that blocks 2019-nCoV entry using a soluble version of the viral receptor, angiotensin-converting enzyme 2 (ACE2), fused to an immunoglobulin Fc domain, providing a neutralizing antibody with maximal breath to avoid any viral escape, while also helping to recruit the immune system to build lasting immunity. The sequence of the ACE2-Fc protein is provided to investigators, allowing its possible use in recombinant protein expression systems to start producing drug today to treat patients under compassionate use, while formal clinical trials are later undertaken. Such a treatment could help infected patients before a protective vaccine is developed and widely available in the coming months to year(s).
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http://dx.doi.org/10.12688/f1000research.22211.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029759PMC
March 2020

Isohemagglutinin titering performed on an automated solid-phase and hemagglutinin-based analyzer is comparable to results obtained by manual gel testing.

Transfusion 2020 03 20;60(3):628-636. Epub 2020 Jan 20.

Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland.

Background: Isohemagglutinins (anti-A and anti-B) mediate hemolytic transfusion reactions, antibody-mediated rejection of solid-organ transplants, and delayed engraftment after stem cell transplant. However, quantification of isohemagglutinins is often labor intensive and operator dependent, limiting availability and interfacility comparisons. We evaluated an automated, solid-phase and agglutination-based antibody titer platform versus manual gel testing.

Study Design And Methods: Plasma samples were obtained from 54 randomly selected patients. Titers were determined by our laboratory's standard assay (manual dilution followed by manual gel testing) and were compared to results obtained on a fully automated blood bank analyzer (Galileo NEO, Immucor). The analyzer determined immunoglobulin G (IgG) antibodies using solid-phase and immunoglobulin M (IgM) antibodies by direct hemagglutination.

Results: Isohemagglutinin titers obtained by manual gel versus the automated assay generally (>80%) agreed within one doubling dilution, and always (100%) agreed within two dilutions. Among O samples, the gel titer and the highest titer obtained with the automated assay (either IgG or IgM) were similar in paired, nonparametric analysis (p = 0.06 for anti-A; p = 0.13 for anti-B). Gel titers from group A and group B patients were slightly higher than the highest titer obtained using the automated assay (p = 0.04 for group A; p = 0.009 for group B), although these differences were within the accepted error of measurement.

Conclusion: Manual and automated methodologies yielded similar isohemagglutinin titers. Separate quantification of IgM and IgG isohemagglutinins via automated titration may yield additional insight into hemolysis, graft survival after ABO-incompatible transplantation, and red blood cell engraftment after ABO-incompatible stem cell transplant.
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http://dx.doi.org/10.1111/trf.15671DOI Listing
March 2020

Corynebacteria as a cause of pulmonary infection: a case series and literature review.

Pneumonia (Nathan) 2018 5;10:10. Epub 2018 Oct 5.

1Baylor College of Medicine, Houston, TX 77030 USA.

Background: In most cases of community-acquired pneumonia (CAP), an etiologic agent is not determined; the most common report from the microbiological evaluation of sputum cites "normal respiratory flora." Non-diphtheria spp. a component of this flora, is commonly viewed as a contaminant, but it may be the cause of pneumonia and the frequency with which it causes CAP may be underestimated.

Case Presentations: This report present 3 cases of CAP in which spp. was clearly the predominant isolate; identification was confirmed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Two cases were caused by and one by Two patients had a tracheostomy and one was on hemodialysis. Patients who received an appropriate antibiotic responded well.

Conclusion: When identified as the predominant isolate in sputum from a patient with CAP, spp. should be considered as a potential cause of the infection. In cases with patients who have compromised airway clearance or who are immunocompromised, microaspiration may be responsible. While some spp. are suspectible to antibiotics usually prescribed for CAP, others are susceptible only to vancomycin or aminoglycosides. Vancomycin is thus the appropriate empiric antibiotic, pending speciation and susceptibility test results. The number of reported cases with result of antibiotic susceptibility testing, however, remains limited, and further investigation is needed. Non-diphtheria spp. represent a noteworthy clinical cause of pneumonia. Identification by Gram stain and as a predominant organism on culture demands careful consideration for management.
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http://dx.doi.org/10.1186/s41479-018-0054-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173903PMC
October 2018

Strategies for enhancing adoptive T-cell immunotherapy against solid tumors using engineered cytokine signaling and other modalities.

Expert Opin Biol Ther 2018 06 14;18(6):653-664. Epub 2018 May 14.

a Center for Cell and Gene Therapy, Texas Children's Hospital , Houston Methodist Hospital, and Baylor College of Medicine , Houston , Texas , USA.

Introduction: Cancer therapy has been transformed by the demonstration that tumor-specific T-cells can eliminate tumor cells in a clinical setting with minimal long-term toxicity. However, significant success in the treatment of leukemia and lymphoma with T-cells using native receptors or redirected with chimeric antigen receptors (CARs) has not been recapitulated in the treatment of solid tumors. This lack of success is likely related to the paucity of costimulatory and cytokine signaling available in solid tumors, in addition to a range of inhibitory mechanisms.

Areas Covered: We summarize the latest developments in engineered T-cell immunotherapy, describe the limitations of these approaches in treating solid tumors, and finally highlight several strategies that may be useful in mediating solid tumor responses in the future, while also ensuring safety of engineered cells.

Expert Opinion: CAR-T therapies require further engineering to achieve their potential against solid tumors. Facilitating cytokine signaling in CAR T-cells appears to be essential in achieving better responses. However, the engineering of T-cells with potentially unchecked proliferation and potency raises the question of whether the simultaneous combination of enhancements will prove safe, necessitating continued advancements in regulating CAR-T activity at the tumor site and methods to safely switch off these engineered cells.
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http://dx.doi.org/10.1080/14712598.2018.1473368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6084433PMC
June 2018

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

Cytotherapy 2018 05 6;20(5):697-705. Epub 2018 Apr 6.

Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, Texas, USA; Center for Stem Cells and Regenerative Medicine, Baylor College of Medicine, Houston, Texas, USA; Translational Biology and Molecular Medicine Program, Baylor College of Medicine, Houston, Texas, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA; Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas, USA. Electronic address:

Background: Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection.

Methods: We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human liver chimeric mice.

Results: HBsAg-CAR T cells recognized HBV-positive cell lines and HBsAg particles in vitro as judged by cytokine production. However, HBsAg-CAR T cells did not kill HBV-positive cell lines in cytotoxicity assays. Adoptive transfer of HBsAg-CAR T cells into HBV-infected humanized mice resulted in accumulation within the liver and a significant decrease in plasma HBsAg and HBV-DNA levels compared with control mice. Notably, the fraction of HBV core-positive hepatocytes among total human hepatocytes was greatly reduced after HBsAg-CAR T cell treatment, pointing to noncytopathic viral clearance. In agreement, changes in surrogate human plasma albumin levels were not significantly different between treatment and control groups.

Conclusions: HBsAg-CAR T cells have anti-HBV activity in an authentic preclinical HBV infection model. Our results warrant further preclinical exploration of HBsAg-CAR T cells as immunotherapy for HBV.
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http://dx.doi.org/10.1016/j.jcyt.2018.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038120PMC
May 2018

In Situ Liver Expression of HBsAg/CD3-Bispecific Antibodies for HBV Immunotherapy.

Mol Ther Methods Clin Dev 2017 Dec 31;7:32-41. Epub 2017 Aug 31.

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA.

Current therapies against hepatitis B virus (HBV) do not reliably cure chronic infection, necessitating new therapeutic approaches. The T cell response can clear HBV during acute infection, and the adoptive transfer of antiviral T cells during bone marrow transplantation can cure patients of chronic HBV infection. To redirect T cells to HBV-infected hepatocytes, we delivered plasmids encoding bispecific antibodies directed against the viral surface antigen (HBsAg) and CD3, expressed on almost all T cells, directly into the liver using hydrodynamic tail vein injection. We found a significant reduction in HBV-driven reporter gene expression (184-fold) in a mouse model of acute infection, which was 30-fold lower than an antibody only recognizing HBsAg. While bispecific antibodies triggered, in part, antigen-independent T cell activation, antibody production within hepatocytes was non-cytotoxic. We next tested the bispecific antibodies in a different HBV mouse model, which closely mimics the transcriptional template for HBV, covalently closed circular DNA (cccDNA). We found that the antiviral effect was noncytopathic, mediating a 495-fold reduction in HBsAg levels at day 4. At day 33, bispecific antibody-treated mice exhibited 35-fold higher host HBsAg immunoglobulin G (IgG) antibody production versus untreated groups. Thus, gene therapy with HBsAg/CD3-bispecific antibodies represents a promising therapeutic strategy for patients with HBV.
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http://dx.doi.org/10.1016/j.omtm.2017.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626922PMC
December 2017

Constitutive Signaling from an Engineered IL7 Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T Cells.

Cancer Discov 2017 11 22;7(11):1238-1247. Epub 2017 Aug 22.

Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, and Baylor College of Medicine, Houston, Texas.

Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but is associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Coexpressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and antitumor activity during repeated exposure to tumor cells, without T-cell dysfunction or autonomous T-cell growth. Furthermore, C7R-coexpressing CAR T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer. The constitutively signaling C7R system developed here delivers potent IL7 stimulation to CAR T cells, increasing their persistence and antitumor activity against multiple preclinical tumor models, supporting its clinical development. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669830PMC
November 2017

In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides.

J Hepatol 2016 Apr 30;64(4):781-9. Epub 2015 Nov 30.

The Scripps Research Institute, La Jolla, CA, USA. Electronic address:

Background & Aims: Current treatment of chronic hepatitis B virus infection (CHB) includes interferon and nucleos(t)ide analogues, which generally do not reduce HBV surface antigen (HBsAg) production, a constellation that is associated with poor prognosis of CHB. Here we evaluated the efficacy of an antisense approach using antisense oligonucleotide (ASO) technology already in clinical use for liver targeted therapy to specifically inhibit HBsAg production and viremia in a preclinical setting.

Methods: A lead ASO was identified and characterized in vitro and subsequently tested for efficacy in vivo and in vitro using HBV transgenic and hydrodynamic transfection mouse and a cell culture HBV infection model, respectively.

Results: ASO treatment decreased serum HBsAg levels ⩾2 logs in a dose and time-dependent manner; HBsAg decreased 2 logs in a week and returned to baseline 4 weeks after a single ASO injection. ASO treatment effectively reduced HBsAg in combination with entecavir, while the nucleoside analogue alone did not. ASO treatment has pan-genotypic antiviral activity in the hydrodynamic transfection system. Finally, cccDNA-driven HBV gene expression is ASO sensitive in HBV infected cells in vitro.

Conclusion: Our results demonstrate in a preclinical setting the efficacy of an antisense approach against HBV by efficiently reducing serum HBsAg (as well as viremia) across different genotypes alone or in combination with standard nucleoside therapy. Since the applied antisense technology is already in clinical use, a lead compound can be rapidly validated in a clinical setting and thus, constitutes a novel therapeutic approach targeting chronic HBV infection.
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http://dx.doi.org/10.1016/j.jhep.2015.11.032DOI Listing
April 2016

Development and rescue of human familial hypercholesterolaemia in a xenograft mouse model.

Nat Commun 2015 Jun 17;6:7339. Epub 2015 Jun 17.

Center for Cell and Gene Therapy, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Diseases of lipid metabolism are a major cause of human morbidity, but no animal model entirely recapitulates human lipoprotein metabolism. Here we develop a xenograft mouse model using hepatocytes from a patient with familial hypercholesterolaemia caused by loss-of-function mutations in the low-density lipoprotein receptor (LDLR). Like familial hypercholesterolaemia patients, our familial hypercholesterolaemia liver chimeric mice develop hypercholesterolaemia and a 'humanized' serum profile, including expression of the emerging drug targets cholesteryl ester transfer protein and apolipoprotein (a), for which no genes exist in mice. We go on to replace the missing LDLR in familial hypercholesterolaemia liver chimeric mice using an adeno-associated virus 9-based gene therapy and restore normal lipoprotein profiles after administration of a single dose. Our study marks the first time a human metabolic disease is induced in an experimental animal model by human hepatocyte transplantation and treated by gene therapy. Such xenograft platforms offer the ability to validate human experimental therapies and may foster their rapid translation into the clinic.
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http://dx.doi.org/10.1038/ncomms8339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4557302PMC
June 2015

Clinical outcomes of hepatitis B virus coinfection in a United States cohort of hepatitis C virus-infected patients.

Hepatology 2014 Dec 29;60(6):1871-8. Epub 2014 Oct 29.

Translational Biology and Molecular Medicine Program, Baylor College of Medicine, Houston, TX.

Unlabelled: The effect of hepatitis B virus (HBV) coinfection in patients with hepatitis C virus (HCV) remains unclear. We used the National Veterans Affairs HCV Clinical Case Registry to identify patients with confirmed HCV viremia during 1997-2005. We defined HBV coinfection as a positive test for hepatitis B surface antigen, HBV DNA, or hepatitis B e antigen. We defined cirrhosis and hepatocellular carcinoma (HCC) based on the validated ICD-9 codes and determined mortality through the end of 2009. We performed Cox proportional hazard regression analyses to examine the effect of HBV coinfection stratified by HBV DNA status (positive or negative) on the risk of cirrhosis, HCC, and death adjusting for patients' age, gender, race, human immunodeficiency virus (HIV) infection, alcohol or drug use, Deyo Score, and antiviral treatment. Among 99,548 patients with HCV infection, 1,370 patients (1.4%) had HBV coinfection. Of the coinfected patients, 677 (49.4%) patients had at least one HBV DNA test done and 303 patients (44.7%) tested positive for HBV DNA. The incidence rates of cirrhosis, HCC, and death were significantly higher in patients with HBV coinfection and detectable HBV DNA compared to HCV monoinfection (36.8, 6.9, and 41.7 versus 17.4, 3.6, and 31.4 per 1,000 person-years, respectively; P < 0.05 for all comparisons). After adjustment for demographic, clinical, and treatment factors, patients with detectable HBV DNA had a significantly higher risk for cirrhosis (hazard ratio [HR] = 1.89 95% confidence interval [CI] = 1.46-2.45), HCC (HR = 2.12, 95% CI = 1.26-3.60), and death (HR = 1.62, 95% CI = 1.33-1.99) compared to HCV monoinfected patients. There were no differences in the risk of cirrhosis, HCC, or overall mortality between coinfected patients with undetectable HBV DNA and those with HCV monoinfection (HR = 1.18, 95% CI = 0.90-1.55; 1.54, 95% CI = 0.93-2.56; 1.08, 95% CI = 0.88-1.33, respectively).

Conclusion: We found that while only a small number of HCV patients were coinfected with HBV, patients with documented HBV viremia were at a significantly higher risk for cirrhosis, HCC, and overall death than HCV monoinfected patients. Absence of HBV replication was associated with a clinical course similar to that of HCV monoinfected patients.
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http://dx.doi.org/10.1002/hep.27337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245372PMC
December 2014

Medium-term results of total knee arthroplasty using a medially pivoting implant: a multicenter study.

J Surg Orthop Adv 2010 ;19(4):191-5

Aurora Advanced Health Care, Mequon, WI, USA.

A multicenter study was conducted to determine the durability and performance of a medially pivoting knee prosthesis in total knee arthroplasty (TKA). Between February 1999 and June 2001, 276 patients underwent 298 primary TKAs at five centers. There were 189 patients (204 knees) available for clinical evaluation after surgery, with an average follow-up of 5.4 years (range, 5.0-7.6 years). The mean age at follow-up was 69 years (range, 39-87 years). The posterior cruciate ligament was resected in 65% of the procedures. Knee Society scores (KSS) and radiographs were assessed for patients who returned for follow-up evaluation. Patients unwilling or unable to return were asked their status via telephone. There were a total of five revisions, and 5-year survivorship using Kaplan-Meier analysis was 97.2%. All radiographs exhibited well-fixed implants with no signs of gross migration or pending failure. Preoperative mean KSS and flexion were 33 and 107°, respectively, improving at latest follow-up to 90 and 121°, respectively. The medial-pivot prosthesis resulted in excellent survivorship with good functional results at medium-term follow-up.
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February 2011
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