Browsing by Subject "gene therapy"
Now showing 1 - 10 of 19
Results Per Page
Sort Options
Item Adenoviral-delivered HE4-HSV-tk sensitizes ovarian cancer cells to ganciclovir(Gene Therapy Press, 2013) Rawlinson, Jennifer W.; Vaden, Kiara; Hunsaker, Joseph; Miller, David F.; Nephew, Kenneth P.; Department of Cellular & Integrative Physiology, IU School of MedicineOvarian cancer (OC) is most often contained within the peritoneal cavity, making it an ideal disease for adenoviral-delivered gene therapies. In effort to develop a safe and effective gene therapy for OC, we created a replication deficient adenovirus bearing the herpes simplex thymidine kinase (HSV-tk) gene under direction of the tumor specific promoter human epididymis protein 4 (HE4). The purpose of this study was to investigate the ability of our adenoviral construct to transduce OC cells in vitro and mediate transgene expression of HSV-tk, thereby sensitizing OC to the pro-drug ganciclovir. Cisplatin-sensitive (CS) and -resistant (CR) A2780 OC cells, infected with virus for 6 hours at 100, 500, and 1000 multiplicity of infection followed by ganciclovir treatment every other day for 5 days, were assayed for cell viability. Adenoviral-mediated transgene expression increased with increasing amounts of virus and peaked at 48 hours after transduction in both A2780-CS and -CR. Unexpectedly, ganciclovir alone was slightly toxic to both A2780 cell lines (IC50 of 234.9 μg/mL and 257.2 μg/mL in A2780-CS and -CR, respectively). Transduction with ADV-HE4-HSV-tk followed by ganciclovir treatment increased (P<0.05) cell killing up to ten-fold, lowering the IC50 to 23.9 μg/mL and 32.6 μg/mL in A2780-CS and -CR, respectively, at 1000 multiplicity of infection. The results support the potential use of this approach as a gene therapy for OC, a disease that accounts for more deaths than any other cancer of the female reproductive system.Item B Cell Depletion Eliminates FVIII Memory B Cells and Enhances AAV8-coF8 Immune Tolerance Induction When Combined With Rapamycin(Frontiers, 2020-06-24) Biswas, Moanaro; Palaschak, Brett; Kumar, Sandeep R. P.; Rana, Jyoti; Markusic, David M.; Pediatrics, School of MedicineHemophilia A is an inherited coagulation disorder resulting in the loss of functional clotting factor VIII (FVIII). Presently, the most effective treatment is prophylactic protein replacement therapy. However, this requires frequent life-long intravenous infusions of plasma derived or recombinant clotting factors and is not a cure. A major complication is the development of inhibitory antibodies that nullify the replacement factor. Immune tolerance induction (ITI) therapy to reverse inhibitors can last from months to years, requires daily or every other day infusions of supraphysiological levels of FVIII and is effective in only up to 70% of hemophilia A patients. Preclinical and recent clinical studies have shown that gene replacement therapy with AAV vectors can effectively cure hemophilia A patients. However, it is unclear how hemophilia patients with high risk inhibitor F8 mutations or with established inhibitors will respond to gene therapy, as these patients have been excluded from ongoing clinical trials. AAV8-coF8 gene transfer in naïve BALB/c-F8e16−/Y mice (BALB/c-HA) results in anti-FVIII IgG1 inhibitors following gene transfer, which can be prevented by transient immune modulation with anti-mCD20 (18B12) and oral rapamycin. We investigated if we could improve ITI in inhibitor positive mice by combining anti-mCD20 and rapamycin with AAV8-coF8 gene therapy. Our hypothesis was that continuous expression of FVIII protein from gene transfer compared to transient FVIII from weekly protein therapy, would enhance regulatory T cell induction and promote deletion of FVIII reactive B cells, following reconstitution. Mice that received anti-CD20 had a sharp decline in inhibitors, which corresponded to FVIII memory B (Bmem) cell deletion. Importantly, only mice receiving both anti-mCD20 and rapamycin failed to increase inhibitors following rechallenge with intravenous FVIII protein therapy. Our data show that B and T cell immune modulation complements AAV8-coF8 gene therapy in naïve and inhibitor positive hemophilia A mice and suggest that such protocols should be considered for AAV gene therapy in high risk or inhibitor positive hemophilia patients.Item Cellular stress and coagulation factor production: When more isn’t necessarily better(Elsevier, 2023-12) Chen, Zhouji; Herzog, Roland W.; Kaufman, Randal J.; Pediatrics, School of MedicineRemarkably, it has been 40 years since the isolation of the 2 genes involved in hemophilia A (HA) and hemophilia B (HB), encoding clotting factor (F) VIII (FVIII) and FIX, respectively. Over the years, these advances led to the development of purified recombinant protein factors that are free of contaminating viruses from human pooled plasma for hemophilia treatments, reducing the morbidity and mortality previously associated with human plasma-derived clotting factors. These discoveries also paved the way for modified factors that have increased plasma half-lives. Importantly, more recent advances have led to the development and Food and Drug Administration approval of a hepatocyte-targeted, adeno-associated viral vector-mediated gene transfer approach for HA and HB. However, major concerns regarding the durability and safety of HA gene therapy remain to be resolved. Compared with FIX, FVIII is a much larger protein that is prone to misfolding and aggregation in the endoplasmic reticulum and is poorly secreted by the mammalian cells. Due to the constraint of the packaging capacity of adeno-associated viral vector, B-domain deleted FVIII rather than the full-length protein is used for HA gene therapy. Like full-length FVIII, B-domain deleted FVIII misfolds and is inefficiently secreted. Its expression in hepatocytes activates the cellular unfolded protein response, which is deleterious for hepatocyte function and survival and has the potential to drive hepatocellular carcinoma. This review is focused on our current understanding of factors limiting FVIII secretion and the potential pathophysiological consequences upon expression in hepatocytes.Item CERE-120 Prevents Irradiation-Induced Hypofunction and Restores Immune Homeostasis in Porcine Salivary Glands(Elsevier, 2020-09-11) Lombaert, Isabelle M. A.; Patel, Vaishali N.; Jones, Christina E.; Villier, Derrick C.; Canada, Ashley E.; Moore, Matthew R.; Berenstein, Elsa; Zheng, Changyu; Goldsmith, Corinne M.; Chorini, John A.; Martin, Daniel; Zourelias, Lee; Trombetta, Mark G.; Edwards, Paul C.; Meyer, Kathleen; Ando, Dale; Passineau, Michael J.; Hoffman, Matthew P.; Oral Pathology, Medicine and Radiology, School of DentistrySalivary gland hypofunction causes significant morbidity and loss of quality of life for head and neck cancer patients treated with radiotherapy. Preventing hypofunction is an unmet therapeutic need. We used an adeno-associated virus serotype 2 (AAV2) vector expressing the human neurotrophic factor neurturin (CERE-120) to treat murine submandibular glands either pre- or post-irradiation (IR). Treatment with CERE-120 pre-IR, not post-IR, prevented hypofunction. RNA sequencing (RNA-seq) analysis showed reduced gene expression associated with fibrosis and the innate and humoral immune responses. We then used a minipig model with CERE-120 treatment pre-IR and also compared outcomes of the contralateral non-IR gland. Analysis of gene expression, morphology, and immunostaining showed reduced IR-related immune responses and improved secretory mechanisms. CERE-120 prevented IR-induced hypofunction and restored immune homeostasis, and there was a coordinated contralateral gland response to either damage or treatment. CERE-120 gene therapy is a potential treatment for head and neck cancer patients to influence communication among neuronal, immune, and epithelial cells to prevent IR-induced salivary hypofunction and restore immune homeostasis.Item Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion(Liebert, 2023-04) Rana, Jyoti; Marsic, Damien; Zou, Chenhui; Muñoz-Melero, Maite; Li, Xin; Kondratov, Oleksandr; Li, Ning; de Jong, Ype P.; Zolotukhin, Sergei; Biswas, Moanaro; Pediatrics, School of MedicineCapsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.Item Decision Making in Fertility Preservation Prior to Pursuing Curative Treatments for Sickle Cell Disease(2023-03-24) Collins, Angela J.; Noel, Josey; Abraham, Olivia; Hornberger, Sydney; Rahim, Mahvish Q.; Jacob, Seethal A.; Saraf, Amanda J.AUTHORS: Angela Collins, MPH(1), Josey Noel(1), Olivia Abraham(1), Sydney Hornberger(1), Mahvish Rahim MD, MBA, MSCR(1,2), Seethal Jacob MD, MS, FAAP(1,2), Amanda Saraf DO(1,2). AFFILIATIONS: (1) Indiana University School of Medicine, Indianapolis, IN. (2) Department of Pediatrics, Riley Hospital for Children, Indianapolis, IN. ABSTRACT: RELEVANT BACKGROUND: Sickle cell disease (SCD) is one of the most commonly inherited hemoglobinopathies, often well controlled on Hydroxyurea (HU). Curative therapy options exist with stem cell transplant (SCT) and gene therapy. While both the underlying condition and routine therapy such as HU is thought to impact fertility, the chemotherapy used for both SCT and gene therapy can result in permanent sterility. Infertility can have a negative impact on long-term measures of quality of life. As a result, fertility preservation ought to be offered to all patients with SCD planning for curative treatment. Ovarian tissue cryopreservation and mature oocyte or embryo cryopreservation are fertility preservation options available for pre and postpubescent females respectively. Testicular tissue cryopreservation (TTC) is an experimental option for prepubescent males and sperm cryopreservation is utilized for postpubescent males. CASE DESCRIPTION: We present three cases of patients with SCD who pursued fertility preservation prior to receiving curative therapy with a myeloablative preparative regimen. Patient 1 is a prepubescent 8-year-old male with SCD controlled with HU who opted for TTC as fertility preservation prior to receiving a matched sibling SCT. Patient 2 is a 13-year-old male with SCD controlled with HU who opted for TTC following a failed sperm banking attempt prior to haploidentical SCT. Patient 3 is an 18-year-old female with SCD controlled with HU and Voxelator who opted to have eggs harvested prior to gene therapy. CLINICAL SIGNIFICANCE: As highlighted by these cases, continued research on safe and effective fertility preservation as well as counseling about both the impact of the underlying disease on fertility and treatment-related fertility risks is imperative to improve long-term quality of life measures. CONCLUSION: These patients demonstrate a need for further emphasis on fertility risk counseling in this patient population and ensuring that discussions regarding preservation options is standard of practice at every institution.Item Ectopic clotting factor VIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma(Elsevier, 2022-12-07) Kapelanski-Lamoureux, Audrey; Chen, Zhouji; Gao, Zu-Hua; Deng, Ruishu; Lazaris, Anthoula; Lebeaupin, Cynthia; Giles, Lisa; Malhotra, Jyoti; Yong, Jing; Zou, Chenhui; de Jong, Ype P.; Metrakos, Peter; Herzog, Roland W.; Kaufman, Randal J.; Pediatrics, School of MedicineHemophilia A gene therapy targets hepatocytes to express B domain deleted (BDD) clotting factor VIII (FVIII) to permit viral encapsidation. Since BDD is prone to misfolding in the endoplasmic reticulum (ER) and ER protein misfolding in hepatocytes followed by high-fat diet (HFD) can cause hepatocellular carcinoma (HCC), we studied how FVIII misfolding impacts HCC development using hepatocyte DNA delivery to express three proteins from the same parental vector: (1) well-folded cytosolic dihydrofolate reductase (DHFR); (2) BDD-FVIII, which is prone to misfolding in the ER; and (3) N6-FVIII, which folds more efficiently than BDD-FVIII. One week after DNA delivery, when FVIII expression was undetectable, mice were fed HFD for 65 weeks. Remarkably, all mice that received BDD-FVIII vector developed liver tumors, whereas only 58% of mice that received N6 and no mice that received DHFR vector developed liver tumors, suggesting that the degree of protein misfolding in the ER increases predisposition to HCC in the context of an HFD and in the absence of viral transduction. Our findings raise concerns of ectopic BDD-FVIII expression in hepatocytes in the clinic, which poses risks independent of viral vector integration. Limited expression per hepatocyte and/or use of proteins that avoid misfolding may enhance safety.Item Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity(Elsevier, 2020-10) Biswas, Moanaro; Marsic, Damien; Li, Ning; Zou, Chenhui; Gonzalez-Aseguinolaza, Gloria; Zolotukhin, Irene; Kumar, Sandeep R.P.; Rana, Jyoti; Butterfield, John S.S.; Kondratov, Oleksandr; de Jong, Ype P.; Herzog, Roland W.; Zolotukhin, Sergei; Pediatrics, School of MedicineLimitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.Item ERT Degrades Gene Therapy for Storage Disorder(Elsevier, 2019-06-13) Markusic, David Michael; Pediatrics, School of MedicineItem Experimental Variables that Affect Human Hepatocyte AAV Transduction in Liver Chimeric Mice(Elsevier, 2020-09-11) Zou, Chenhui; Vercauteren, Koen O. A.; Michailidis, Eleftherios; Kabbani, Mohammad; Zoluthkin, Irene; Quirk, Corrine; Chiriboga, Luis; Yazicioglu, Mustafa; Anguela, Xavier M.; Meuleman, Philip; High, Katherine A.; Herzog, Roland W.; Jong, Ype P. de; Pediatrics, School of MedicineAdeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah−/−) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah−/− chimera model, attention to which may improve reproducibility between findings from different laboratories.